What was explored on CYTOO's micropatterns?

carcinoma cells

apoptosis

apoptosis, golgi

carcinoma cells

integrins, myosin, forces

circadian clock, primary cells

Filamin A

vesicular trafficking

Immunology, Buruli ulcer

mechanotransduction, cardiomyocytes

cytoskeleton

cell shape

Cytoskeleton, protein

nucleus positioning

extracellular matrix, cell-cell junction

cardiomyocytes,

cell adhesion

Spindle position

spindle position

spindle orientation

spindle position

dynein

spindle position, centriole

microcephaly proteins

Spindle position

Spindle position

cell morphology

genomics

macrophage tumor

cell pairing

cell streaming

endothelial cells

focal adhesions

Kif1C, cell tails

golgi, centrosome

ciliogenesis

World Cell Race

CD81, Rac

RPGR, primary cilia, actin

cell shape, cell contractility

primary cilia

protein localization protein movement

stress fibers, Ca2+, myosin

dysferlin

gene transfer

lysosomes

microtubules

nucleus, cytoskeleton

force transmission

YAP/TAZ, extracellular matrix, mechanotransduction, cell perception

procollagen trafficking

osteoblasts

mitochondrial dynamics, mitochondrial networks

mitochondria, microtubule, cell shape

epithelial cells, lumen, cell contractility

cell confinement, centrosome, lumen, epithelial morphogenesis

YAP/TAZ, cell shape, cell proliferation

Tissue engineering

3D cell culture, apical lumen

epithelial morphogenesis

Organelle map, high content screening, high throughput screening

quantitative analysis

toxicity

cell morphology

quantitative comparison

Organelle Map

endomembrane organization

cell division, cell adhesion

cell adhesion, cytoskeleton

Cell adhesion

Sharma V, Beaty B, Patsialou A, et al. Reconstitution of in vivo macrophage-tumor cell pairing and streaming motility on one-dimensional micro-patterned substrates. IntraVital. 2012;1(1):77–85.

Kitagawa D, Kohlmaier G, Keller D, et al. Spindle positioning in human cells relies on proper centriole formation and on the microcephaly proteins CPAP and STIL. J. Cell. Sci. 2011;124(22):3884–3893.

Kotak S, Busso C, Gönczy P. Cortical dynein is critical for proper spindle positioning in human cells. J Cell Biol. 2012;199(1):97–110.

Theisen U, Straube E, Straube A. Directional persistence of migrating cells requires Kif1C-mediated stabilization of trailing adhesions. Dev. Cell. 2012;23(6):1153–1166.

Devallière J, Chatelais M, Fitau J, et al. LNK (SH2B3) is a key regulator of integrin signaling in endothelial cells and targets -parvin to control cell adhesion and migration. FASEB J. 2012;26(6):2592–2606.

Kiyomitsu T, Cheeseman IM. Chromosome- and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation. Nat. Cell Biol. 2012;14(3):311–317.

Hurtado L, Caballero C, Gavilan MP, Cardenas J, Bornens M, Rios RM. Disconnecting the Golgi ribbon from the centrosome prevents directional cell migration and ciliogenesis. J. Cell Biol. 2011;193(5):917–933.

Stiles JM, Pham R, Rowntree RK, et al. Morphological restriction of human coronary artery endothelial cells substantially impacts global gene expression patterns. FEBS J. 2013; June 27 [epub ahead of print].

Samora CP, Mogessie B, Conway L, Ross JL, Straube A, McAinsh AD. MAP4 and CLASP1 operate as a safety mechanism to maintain a stable spindle position in mitosis. Nat. Cell Biol. 2011;13(9):1040–1050.

Kotak S, Gönczy P. Mechanisms of spindle positioning: cortical force generators in the limelight. Curr Opin Cell Biol. 2013; Aug 16 [epub ahead of print].

Kiyomitsu T, Cheeseman IM. Cortical Dynein and Asymmetric Membrane Elongation Coordinately Position the Spindle in Anaphase. Cell. 2013;154(2):391–402.

Abstract: Correct spindle positioning is fundamental for proper cell division during development and in stem cell lineages. Dynein and an evolutionarily conserved ternary complex (nuclear mitotic apparatus protein [NuMA]–LGN–G in human cells and LIN-5–GPR-1/2–G in Caenorhabditis elegans) are required for correct spindle positioning, but their relationship remains incompletely understood. By analyzing fixed specimens and conducting live-imaging experiments, we uncovered that appropriate levels of ternary complex components are critical for dynein-dependent spindle positioning in HeLa cells and C. elegans embryos. Moreover, using mutant versions of G in both systems, we established that dynein acts at the membrane to direct spindle positioning. Importantly, we identified a region within NuMA that mediates association with dynein. By using this region to target dynein to the plasma membrane, we demonstrated that the mere presence of dynein at that location is sufficient to direct spindle positioning in HeLa cells. Overall, we propose a model in which the ternary complex serves to anchor dynein at the plasma membrane to ensure correct spindle positioning.

Abstract: Directional cell migration requires the establishment and maintenance of long-term differences in structure and function between the front and back of a cell. Here, we show that the microtubule motor Kif1C contributes to persistent cell migration primarily through stabilization of an extended cell rear. Kif1C-mediated transport of 51-integrins is required for the proper maturation of trailing focal adhesions and resistance to tail retraction. Tail retraction precedes and induces changes in migration direction. Stabilization of cell tails through inhibition of myosin II activity suppresses the Kif1C depletion phenotype and results in longer-lived tails and higher directional stability of migrating cells. Taken together, these findings indicate that the maintenance of an extended, tense cell tail facilitates directional migration. We propose a rear drag mechanism for directional persistence of migration whereby the counterforce originating from a well-anchored tail serves to maintain directionality of the force-generating leading edge of the cell.

Abstract: Correct positioning of the spindle governs placement of the cytokinesis furrow and thus plays a crucial role in the partitioning of fate determinants and the disposition of daughter cells in a tissue. Converging evidence indicates that spindle positioning is often dictated by interactions between the plus-end of astral microtubules that emanate from the spindle poles and an evolutionary conserved cortical machinery that serves to pull on them. At the heart of this machinery lies a ternary complex (LIN-5/GPR-1/2/G alpha in Caenorhabditis elegans and NuMA/LGN/G alpha i in Homo sapiens) that promotes the presence of the motor protein dynein at the cell cortex. In this review, we discuss how the above components contribute to spindle positioning and how the underlying mechanisms are precisely regulated to ensure the proper execution of this crucial process in metazoan organisms

Abstract: Alterations in cell shape have been shown to modulate chromatin condensation and cell lineage specification, however the mechanisms controlling these processes are largely unknown. Because endothelial cells experience cyclic mechanical changes from blood flow during normal physiological processes and disrupted mechanical changes due to abnormal blood flow, cell shape deformation, and loss of polarization during coronary artery disease, we sought to elucidate how morphological restriction affects global gene expression patterns. Human coronary artery endothelial cells (HCAECs) were cultured on spatially defined adhesive micropatterns forcing them to conform to unique cellular morphologies differing in cellular polarization and angularity. We utilized pattern recognition algorithms and statistical analysis to validate the cytoskeletal pattern reproducibility and uniqueness of each micropattern, and performed microarray analysis on normal shaped and micropatterned HCAECs to determine how constrained cellular morphology affects gene expression patterns. Analysis of the data revealed that forcing HCAECs to conform to geometrically defined shapes significantly affects their global transcription patterns compared to non-restricted shapes. Interestingly, gene expression patterns were altered in response to morphological restriction in general, but were consistent regardless of the particular shape the cells conformed to. These data suggest that the ability of HCAECs to spread, but not necessarily their particular morphology, dictates their genomics patterns.

Abstract: Patients with MCPH (autosomal recessive primary microcephaly) exhibit impaired brain development, presumably due to the compromised function of neuronal progenitors. Seven MCPH loci have been identified, including one that encodes centrosome protein 4.1 associated protein (CPAP; also known as centromere protein J, CENPJ). CPAP is a large coiled-coil protein enriched at the centrosome, a structure that comprises two centrioles and surrounding pericentriolar material (PCM). CPAP depletion impairs centriole formation, whereas CPAP overexpression results in overly long centrioles. The mechanisms by which CPAP MCPH patient mutations affect brain development are not clear. Here, we identify CPAP protein domains crucial for its centriolar localization, as well as for the elongation and the formation of centrioles. Furthermore, we demonstrate that conditions that resemble CPAP MCPH patient mutations compromise centriole formation in tissue culture cells. Using adhesive micropatterns, we reveal that such defects correlate with a randomization of spindle position. Moreover, we demonstrate that the MCPH protein SCL/TAL1 interrupting locus (STIL) is also essential for centriole formation and for proper spindle position. Our findings are compatible with the notion that mutations in CPAP and STIL cause MCPH because of aberrant spindle positioning in progenitor cells during brain development.

Abstract: In mammary tumors, intravital imaging techniques have uncovered an essential role for macrophages during tumor cell invasion and metastasis mediated by an epidermal growth factor (EGF) / colony stimulating factor-1 (CSF-1) paracrine loop. It was previously demonstrated that mammary tumors in mice derived from rat carcinoma cells (MTLn3) exhibited high velocity migration on extracellular matrix (ECM) fibers. These cells form paracrine loop-dependent linear assemblies of alternating host macrophages and tumor cells known as “streams.” Here, we confirm by intravital imaging that similar streams form in close association with ECM fibers in a highly metastatic patient-derived orthotopic mammary tumor (TN1). To understand the in vivo cell motility behaviors observed in streams, an in vitro model of fibrillar tumor ECM utilizing adhesive 1D micropatterned substrates was developed. MTLn3 cells on 1D fibronectin or type I collagen substrates migrated with higher velocity than on 2D substrates and displayed enhanced lamellipodial protrusion and increased motility upon local interaction and pairing with bone marrow-derived macrophages (BMMs). Inhibitors of EGF or CSF-1 signaling disrupted this interaction and reduced tumor cell velocity and protrusion, validating the requirement for an intact paracrine loop. Both TN1 and MTLn3 cells in the presence of BMMs were capable of co-assembling into linear arrays of alternating tumor cells and BMMs that resembled streams in vivo, suggesting the stream assembly is cell autonomous and can be reconstituted on 1D substrates. Our results validate the use of 1D micropatterned substrates as a simple and defined approach to study fibrillar ECM-dependent cell pairing, migration and relay chemotaxis as a complementary tool to intravital imaging.

Abstract: Correct positioning of the mitotic spindle is critical to establish the correct cell-division plane. Spindle positioning involves capture of astral microtubules and generation of pushing/pulling forces at the cell cortex. Here we show that the tau-related protein MAP4 and the microtubule rescue factor CLASP1 are essential for maintaining spindle position and the correct cell-division axis in human cells. We propose that CLASP1 is required to correctly capture astral microtubules, whereas MAP4 prevents engagement of excess dynein motors, thereby protecting the system from force imbalance. Consistent with this, MAP4 physically interacts with dynein-dynactin in vivo and inhibits dynein-mediated microtubule sliding in vitro. Depletion of MAP4, but not CLASP1, causes spindle misorientation in the vertical plane, demonstrating that force generators are under spatial control. These findings have wide biological importance, because spindle positioning is essential during embryogenesis and stem-cell homeostasi

Abstract: Mammalian cells exhibit a frequent pericentrosomal Golgi ribbon organization. In this paper, we show that two AKAP450 N-terminal fragments, both containing the Golgi-binding GM130-interacting domain of AKAP450, dissociated endogenous AKAP450 from the Golgi and inhibited microtubule (MT) nucleation at the Golgi without interfering with centrosomal activity. These two fragments had, however, strikingly different effects on both Golgi apparatus (GA) integrity and positioning, whereas the short fragment induced GA circularization and ribbon fragmentation, the large construct that encompasses an additional p150glued/MT-binding domain induced separation of the Golgi ribbon from the centrosome. These distinct phenotypes arose by specific interference of each fragment with either Golgi-dependent or centrosome-dependent stages of Golgi assembly. We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon. Both features, however, were required for ciliogenesis. We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.

Abstract: Mitotic spindle positioning by cortical pulling forces defines the cell division axis and location, which is critical for proper cell division and development. Although recent work has identified developmental and extrinsic cues that regulate spindle orientation, the contribution of intrinsic signals to spindle positioning and orientation remains unclear. Here, we demonstrate that cortical force generation in human cells is controlled by distinct spindle-pole- and chromosome-derived signals that regulate cytoplasmic dynein localization. First, dynein exhibits a dynamic asymmetric cortical localization that is negatively regulated by spindle-pole proximity, resulting in spindle oscillations to centre the spindle within the cell. We find that this signal comprises the spindle-pole-localized polo-like kinase (Plk1), which regulates dynein localization by controlling the interaction between dynein-dynactin and its upstream cortical targeting factors NuMA and LGN. Second, a chromosome-derived RanGTP gradient restricts the localization of NuMA-LGN to the lateral cell cortex to define and maintain the spindle orientation axis. RanGTP acts in part through the nuclear localization sequence of NuMA to locally alter the ability of NuMA-LGN to associate with the cell cortex in the vicinity of chromosomes. We propose that these chromosome- and spindle-pole-derived gradients generate an intrinsic code to control spindle position and orientation.

Abstract: Focal adhesion (FA) formation and disassembly play an essential role in adherence and migration of endothelial cells. These processes are highly regulated and involve various signaling molecules that are not yet completely identified. Lnk [Src homology 2-B3 (SH2B3)] belongs to a family of SH2-containing proteins with important adaptor functions. In this study, we showed that Lnk distribution follows that of vinculin, localizing Lnk in FAs. Inhibition of Lnk by RNA interference resulted in decreased spreading, whereas sustained expression dramatically increases the number of focal and cell-matrix adhesions. We demonstrated that Lnk expression impairs FA turnover and cell migration and regulates 1-integrin-mediated signaling via Akt and GSK3 phosphorylation. Moreover, the -parvin protein was identified as one of the molecular targets of Lnk responsible for impaired FA dynamics and cell migration. Finally, we established the ILK protein as a new molecular partner for Lnk and proposed a model in which Lnk regulates -parvin expression through its interaction with ILK. Collectively, our results underline the adaptor Lnk as a novel and effective key regulator of integrin-mediated signaling controlling endothelial cell adhesion and migration.

Abstract: Mitotic spindle position defines the cell-cleavage site during cytokinesis. However, the mechanisms that control spindle positioning to generate equal-sized daughter cells remain poorly understood. Here, we demonstrate that two mechanisms act coordinately to center the spindle during anaphase in symmetrically dividing human cells. First, the spindle is positioned directly by the microtubule-based motor dynein, which we demonstrate is targeted to the cell cortex by two distinct pathways: a G alpha i/LGN/NuMA-dependent pathway and a 4.1G/R and NuMA-dependent, anaphase-specific pathway. Second, we find that asymmetric plasma membrane elongation occurs in response to spindle mispositioning to alter the cellular boundaries relative to the spindle. Asymmetric membrane elongation is promoted by chromosome-derived Ran-GTP signals that locally reduce Anillin at the growing cell cortex. In asymmetrically elongating cells, dynein-dependent spindle anchoring at the stationary cell cortex ensures proper spindle positioning. Our results reveal the anaphase-specific spindle centering systems that achieve equal-sized cell division.

"Interestingly, experiments with fibronectin micro-patterns revealed that cells utilize focal adhesions and actin fibers established during interphase to impart spindle positioning during mitosis."

http://www.sciencedirect.com/science/article/pii/S0955067413001221

"Spindle position is strongly linked to the cell-division axis1,13,34. To directly test whether the absence of MAP4or CLASP1 results in divisionplane errors, we observed dividing immortalized primary epithelial hTERT-RPE1 cells on adhesive substrate patterns that promote a single division plane12. In control cells, more than 50% of cell divisions occurred in the direction guided by the extracellular pattern with a precision of 6. Consistent with the spindle-orientation defects reported here, MAP4 or CLASP1 depletion led to wider angular distributions of cell-division axes".

http://www.nature.com/ncb/journal/v13/n9/full/ncb2297.html

"In this investigation, we examined the global gene expression changes that occur when human coronary artery endothelial cells (HCAECs) are shape and spread restricted by micropatterning into reproducibly unique cellular morphologies that are distinctive in polarization, morphological angularity, and actin cytoskeleton patterning."

http://onlinelibrary.wiley.com/doi/10.1111/febs.12410/abstract

"Our work demonstrates a role of the AKAP450–GM130 complex in organizing the Golgi ribbon around the CTR. How this interaction is regulated during the cell cycle and how GA-associated MT nucleation is inhibited at the onset of mitosis to ensure the proper formation of the spindle are interesting questions that deserve attention for the future."

http://jcb.rupress.org/content/193/5/917.long

"We utilized microfabricated chips as a novel assay for analyzing spindle positioning upon depletion of MCPH components or in conditions mimicking the mutations in MCPH patients (...) our findings raise the possibility that different MCPH components, although required for distinct cell biological processes, all affect brain development because their impairment leads to a randomization of spindle position in progenitor cells. In addition, the centrosomal protein Cep152 is also needed for centriole formation and is another MCPH component."

http://jcs.biologists.org/content/124/22/3884.long

"Our in vivo work with human cells is compatible withthese in vitro results because we found that dynein at the plasma membrane is sufficient to direct spindle positioning independently of the ternary complex. It will be interesting to analyze microtubule dynamics in mitotic cells expressing GFP-NuMA (1–705)-CAAX to monitor the influence of plasma membrane dynein on microtubules. These and related approaches should help further clarify the mechanisms by which the presence of dynein at the cell cortex dictates proper spindle positioning in metazoan organisms"

http://jcb.rupress.org/content/199/1/97

"Our work elucidates the functional significance of cell tails and gives mechanistic insight into the role of Kif1C, a transport kinesin, in maintaining the directional persistence of cell migration. Deregulating the coordination of rear adhesion and forward locomotion disturbs cell polarity and is likely to impact physiological processes that depend on persistent directional migration during embryogenesis and immune surveillance."

http://www.cell.com/developmental-cell/retrieve/pii/S1534580712005254

"Our findings suggest that Lnk is a pivotal adaptor in vascular ECs that regulates at least two major signaling pathways (TNF and -integrin) in the endothelium. Finally, the regulatory functions of Lnk in multiple interconnected intracellular signaling pathways highlights its therapeutic potential as a molecular target for the prevention of vascular diseases".

http://www.fasebj.org/content/26/6/2592.abstract

"Defining the mechanisms that control spindle position and orientation is key to understanding cell division. Here, we demonstrated that cortical dynein localization is regulated by two distinct intrinsic signals to correct spindle position and orientation in symmetrically dividing human cells (...) we found that Gi and LGN-C localize to retraction fibres, which have been proposed to dictate spindle orientation on patterned substrates. On the basis of the connections between spindle orientation and tumorigenesis21, this work also has important relevance to studies on cancer progression."

http://www.nature.com/ncb/journal/v14/n3/full/ncb2440.html

"Our 1D micropatterned substrate model more closely approximates the fibrillar nature of the in vivo tumor microenvironment and offers a simple and more appropriate substrate for detailed analyses of cell protrusion, cell-cell pairing and migration than conventional 2D substrates. The data presented here validates the use of micropatterned 1D adhesive substrates to study the fibrillar ECM found within the tumor microenvironment. The composition and geometry of the 1D micropatterned substrate can be easily customized in order to study the role of the ECM during high velocity tumor cell migration, cell-cell pairing and streaming".

http://www.landesbioscience.com/journals/intravital/article/22054/

"Our results reveal that cortical dynein and membrane elongation coordinately control spindle positioning. Both mechanisms are autonomously regulated in response to spindle position and cooperatively center the spindle to achieve an equal-sized cell division".

http://www.sciencedirect.com/science/article/pii/S0092867413007125

Maiuri P, Terriac E, Paul-Gilloteaux P, et al. The first World Cell Race. Curr Biol. 2012;22(17):R673–R675.

Abstract: Motility is a common property of animal cells. Cell motility is required for embryogenesis [1], tissue morphogenesis [2] and the immune response [3] but is also involved in disease processes, such as metastasis of cancer cells [4]. Analysis of cell migration in native tissue in vivo has yet to be fully explored, but motility can be relatively easily studied in vitro in isolated cells. Recent evidence suggests that cells plated in vitro on thin lines of adhesive proteins printed onto culture dishes can recapitulate many features of in vivo migration on collagen fibers [5,6]. However, even with controlled in vitro measurements, the characteristics of motility are diverse and are dependent on the cell type, origin and external cues. One objective of the first World Cell Race was to perform a large-scale comparison of motility across many different adherent cell types under standardized conditions. To achieve a diverse selection, we enlisted the help of many international laboratories, who submitted cells for analysis. The large-scale analysis, made feasible by this competition-oriented collaboration, demonstrated that higher cell speed correlates with the persistence of movement in the same direction irrespective of cell origin.

"Together, the results generated by the first World Cell Race highlight how scientific games involving largescale experiments can lead to the identification of novel and relevant biological processes, which may otherwise escape observation".

http://www.cell.com/current-biology/abstract/S0960-9822%2812%2900877-9

Tejera E, Rocha-Perugini V, López-Martín S, et al. CD81 regulates cell migration through its association with Rac GTPase. Mol Biol Cell. 2012;24(3):261–273.

Abstract: CD81 is a member of the tetraspanin family that has been described to have a key role in cell migration of tumor and immune cells. To unravel the mechanisms of CD81-regulated cell migration, we performed proteomic analyses that revealed an interaction of the tetraspanin C-terminal domain with the small GTPase Rac. Direct interaction was confirmed biochemically. Moreover, microscopy cross-correlation analysis demonstrated the in situ integration of both molecules into the same molecular complex. Pull-down experiments revealed that CD81-Rac interaction was direct and independent of Rac activation status. Knockdown of CD81 resulted in enhanced protrusion rate, altered focal adhesion formation and decreased cell migration, correlating with increased active Rac. Re-expression of wild type CD81, but not its truncated form lacking the C-terminal cytoplasmic domain, rescued these effects. The phenotype of CD81 knockdown cells was mimicked by treatment with a soluble peptide with the C-terminal sequence of the tetraspanin. Our data show that the interaction of Rac with the C-terminal cytoplasmic domain of CD81 is a novel regulatory mechanism of the GTPase activity turnover. Furthermore, they provide a novel mechanism for tetraspanin-dependent regulation of cell motility and open new avenues for tetraspanin-targeted reagents by the use of cell permeable peptides.

Cell infection

Cell contractility

Cell fate

Cell migration

"Our results map the regulatory effect of CD81 to its C-terminal cytoplasmic region, which strongly suggests that the role of CD81 in controlling cell migration is due to its interaction with Rac rather than its lateral association with other membrane receptors in the context of tetraspanin-enriched microdomains. Moreover, our data with cell-permeable peptides with the sequence of CD81 C-terminal domain, suggest they may be an exciting tool to affect tetraspanin-dependent biological processes in vivo, offering a new expectative in the therapeutic value of tetraspanin-targeted reagents for the treatment of malignancies or immunerelated diseases."

http://www.molbiolcell.org/content/early/2012/12/11/mbc.E12-09-0642

Cell division

Guenot M, Racz P. Practical course on “imaging infection: from single molecules to animals”. Microbes Infect. 2012;14(15):1475–1482.

Chopra A, Patel A, Shieh AC, A Janmey P, Kresh JY. a-Catenin Localization and Sarcomere Self-Organization on N-Cadherin Adhesive Patterns Are Myocyte Contractility Driven. PLoS ONE. 2012;7(10):e47592.

Abstract: The N-cadherin (N-cad) complex plays a crucial role in cardiac cell structure and function. Cadherins are adhesion proteins linking adjacent cardiac cells and, like integrin adhesions, are sensitive to force transmission. Forces through these adhesions are capable of eliciting structural and functional changes in myocytes. Compared to integrins, the mechanisms of force transduction through cadherins are less explored. -catenin is a major component of the cadherin-catenin complex, thought to provide a link to the cell actin cytoskeleton. Using N-cad micropatterned substrates in an adhesion constrainment model, the results from this study show that -catenin localizes to regions of highest internal stress in myocytes. This localization suggests that a-catenin acts as an adaptor protein associated with the cadherin mechanosensory apparatus, which is distinct from mechanosensing through integrins. Myosin inhibition in cells bound by integrins to fibronectin-coated patterns disrupts myofibiril organization, whereas on N-cad coated patterns, myosin inhibition leads to better organized myofibrils. This result indicates that the two adhesion systems provide independent mechanisms for regulating myocyte structural organization.

Abstract: A 2 weeklong theoretical and practical course on innovative microscopy in the field of microbial infection was organized in Pretoria, South Africa. Talks from lecturers from such fields as super-resolution microscopy, fluorescence and bioluminescence imaging, high throughput microscopy assays and image analysis were followed by practicals on cutting edge microscopes.

Gakovic M, Shu X, Kasioulis I, Carpanini S, Moraga I, Wright AF. The role of RPGR in cilia formation and actin stability. Hum. Mol. Genet. 2011;20(24):4840–4850.

"The use of single cell micro-patterns provides an effective way to control a cell’s shape and mechanical microenvironment in a reproducible manner. ‘‘Y’’ shaped micropatterns, in particular, provide a unique geometry with three polarized anisotropic ends and an isotropic center (...) our results indicate that cell-cell and cell-ECM adhesion systems provide independent degrees of freedom for tuning cardiac myocyte structural organization."

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0047592

Abstract: Mutations in the retinitis pigmentosa GTPase regulator (RPGR) protein cause one of the most common and severe forms of inherited retinal dystrophy. In spite of numerous studies, the precise function of RPGR remains unclear, as is the mechanism by which RPGR mutations cause retinal degeneration. We have analysed the function of RPGR by RNA interference-mediated translational suppression [knockdown (KD)] using a model cellular system for studying the formation, maintenance and function of primary cilia (human telomerase-immortalized retinal pigmented epithelium 1 cells). We observed that RPGR-deficient cells exhibited reduced numbers of cilia, slower cell cycle progression and impaired attachment to fibronectin, but showed no migration defects in a wound-healing assay. RPGR KD cells showed stronger actin filaments, associated with basal dysregulation of the Akt, Erk1/2, focal adhesion kinase and Src signalling pathways, as well as a 20% reduction in 1-integrin receptors at the cell surface and impaired fibronectin-induced signalling. Stronger actin filaments and impairment of the above signalling pathways suggest a common underlying mechanism for all of the cellular phenotypes observed in RPGR KD cells. Our data underline a novel function for RPGR in cilia formation and in the regulation of actin stress filaments, suggesting that, in the retina, it may regulate nascent photoreceptor disc formation by regulating actin-mediated membrane extension.

Courtesy of Landes Bioscience, reproduced from IntraVital 2012;1(1): 77-85.

"Altogether, our data suggest that while RPGR may associate with a variety of other transport and scaffold proteins en route to the cilium, a major function within the photoreceptor cilium is nascent disc formation, by regulating actin-mediated membrane extension. Since RPGR has been found to interact with Rab8, a small GTPase involved in ciliogenesis and rhodopsin trafficking, it is tempting to hypothesize that RPGR mediates previously reported Rab8-positive vesicle docking to microfilament bundles and/or coordinates vesicle docking with membrane extension."

http://hmg.oxfordjournals.org/content/20/24/4840.long

"3T3 mouse fibroblasts grown on Cy5-fibronektin-coated micropatterns were infected by GFPShigella flexneri or GFP-Listeria monocytogenes (...) The bacteria are capable to form entry sites at the apical and basolateral sites of the challenged patterned cells."

http://www.sciencedirect.com/science/article/pii/S1286457912002602

Read the CYTOO Story "Tumor ECM: the highway to cell"

http://www.cytoo.com/documents/CytooStories_TumorCellMigration_web3_000.pdf

Courtesy of Brad Bryan, Department of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, USA

©Chopra et al. Plos One 2012

Pitaval A, Tseng Q, Bornens M, Théry M. Cell shape and contractility regulate ciliogenesis in cell cycle-arrested cells. J. Cell Biol. 2010;191(2):303–312.

Abstract: In most lineages, cell cycle exit is correlated with the growth of a primary cilium. We analyzed cell cycle exit and ciliogenesis in human retinal cells and found that, contrary to the classical view, not all cells exiting the cell division cycle generate a primary cilium. Using adhesive micropatterns to control individual cell spreading, we demonstrate that cell spatial confinement is a major regulator of ciliogenesis. When spatially confined, cells assemble a contractile actin network along their ventral surface and a protrusive network along their dorsal surface. The nucleus-centrosome axis in confined cells is oriented toward the dorsal surface where the primary cilium is formed. In contrast, highly spread cells assemble mostly contractile actin bundles. The nucleus-centrosome axis of spread cells is oriented toward the ventral surface, where contractility prevented primary cilium growth. These results indicate that cell geometrical confinement affects cell polarity via the modulation of actin network architecture and thereby regulates basal body positioning and primary cilium growth.

Ciliogenesis

"When individual cells were sufficiently spatially confined on adhesive substrate, they could assemble both a contractile ventral domain and a protrusive, ezrin-rich dorsal domain mimicking the apicobasal polarity of epithelial cells. This polarization was further transmitted to the internal cell organization with the nucleus– centrosome axis oriented toward the dorsal surface in a Rho kinase–dependent manner. Under these conditions, most cells assemble a primary cilium".

http://jcb.rupress.org/content/191/2/303.long

Cell-cell junction

Chevrollier A, Cassereau J, Ferré M, et al. Standardized mitochondrial analysis gives new insights into mitochondrial dynamics and OPA1 function. Int. J. Biochem. Cell Biol. 2012;44(6):980–988.

Mitochondrial analysis

Cunningham J, Estrella V, Lloyd M, Gillies R, Frieden BR, Gatenby R. Intracellular Electric Field and pH Optimize Protein Localization and Movement. PLoS ONE. 2012;7(5):e36894.

1. Kresh JY, Chopra A. Intercellular and extracellular mechanotransduction in cardiac myocytes. Pflugers Arch. 2011;462(1):75–87.

2. Chopra A, Patel A, Shieh AC, A Janmey P, Kresh JY. -Catenin Localization and Sarcomere Self-Organization on N-Cadherin Adhesive Patterns Are Myocyte Contractility Driven. PLoS ONE. 2012;7(10):e47592.

Ciliogenesis

1. Gakovic M, Shu X, Kasioulis I, Carpanini S, Moraga I, Wright AF. The role of RPGR in cilia formation and actin stability. Hum. Mol. Genet. 2011;20(24):4840–4850.

2. Pitaval A, Tseng Q, Bornens M, Théry M. Cell shape and contractility regulate ciliogenesis in cell cycle-arrested cells. J. Cell Biol. 2010;191(2):303–312.

Chevrollier A, Cassereau J, Ferré M, et al. Standardized mitochondrial analysis gives new insights into mitochondrial dynamics and OPA1 function. Int. J. Biochem. Cell Biol. 2012;44(6):980–988.

Mitochondrial analysis

Cardiac function

Abstract: Mammalian cell function requires timely and accurate transmission of information from the cell membrane (CM) to the nucleus (N). These pathways have been intensively investigated and many critical components and interactions have been identified. However, the physical forces that control movement of these proteins have received scant attention. Thus, transduction pathways are typically presented schematically with little regard to spatial constraints that might affect the underlying dynamics necessary for protein-protein interactions and molecular movement from the CM to the N. We propose messenger protein localization and movements are highly regulated and governed by Coulomb interactions between: 1. A recently discovered, radially directed E-field from the NM into the CM and 2. Net protein charge determined by its isoelectric point, phosphorylation state, and the cytosolic pH. These interactions, which are widely applied in elecrophoresis, provide a previously unknown mechanism for localization of messenger proteins within the cytoplasm as well as rapid shuttling between the CM and N. Here we show these dynamics optimize the speed, accuracy and efficiency of transduction pathways even allowing measurement of the location and timing of ligand binding at the CM –previously unknown components of intracellular information flow that are, nevertheless, likely necessary for detecting spatial gradients and temporal fluctuations in ligand concentrations within the environment. The model has been applied to the RAF-MEK-ERK pathway and scaffolding protein KSR1 using computer simulations and in-vitro experiments. The computer simulations predicted distinct distributions of phosphorylated and unphosphorylated components of this transduction pathway which were experimentally confirmed in normal breast epithelial cells (HMEC).

Tseng Q, Duchemin-Pelletier E, Deshiere A, et al. Spatial organization of the extracellular matrix regulates cell-cell junction positioning. Proc. Natl. Acad. Sci. U.S.A. 2012;109(5):1506–1511.

"These previously unknown mechanisms provide a mechanism for controlling the optimal localization of messenger proteins within the cytoplasm as well rapid movement from the CM to the NM preserving information on the location and time of ligand binding."

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0036894

Abstract: The organization of cells into epithelium depends on cell interaction with both the extracellular matrix (ECM) and adjacent cells. The role of cell-cell adhesion in the regulation of epithelial topology is well-described. ECM is better known to promote cell migration and provide a structural scaffold for cell anchoring, but its contribution to multicellular morphogenesis is less well-understood. We developed a minimal model system to investigate how ECM affects the spatial organization of intercellular junctions. Fibronectin micropatterns were used to constrain the location of cell-ECM adhesion. We found that ECM affects the degree of stability of intercellular junction positioning and the magnitude of intra- and intercellular forces. Intercellular junctions were permanently displaced, and experienced large perpendicular tensional forces as long as they were positioned close to ECM. They remained stable solely in regions deprived of ECM, where they were submitted to lower tensional forces. The heterogeneity of the spatial organization of ECM induced anisotropic distribution of mechanical constraints in cells, which seemed to adapt their position to minimize both intra- and intercellular forces. These results uncover a morphogenetic role for ECM in the mechanical regulation of cells and intercellular junction positioning.

"Although much attention has been paid to the role of cell–cell interaction in the shaping of epithelia, our results suggest that we should also consider the organizing role of ECM, which, far from being a mere supporting scaffold, plays an instructive role in regulating mechanical forces and orienting multicellular assembly."

http://www.pnas.org/content/109/5/1506.long

Read the CYTOO Story "Restoring cell function on micropatterns: empowering cells to grow a primary cilium"

http://www.cytoo.com/documents/CytooStories_Ciliogenesis.pdf

Badyal SK, Basran J, Bhanji N, et al. Mechanism of the Ca2+-Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA. J Mol Biol. 2011;405(4-3):1004–1026.

Abstract: The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca2+ binds to the EF2 domain of S100A4 with micromolar affinity and that the Kd value for Ca2+ is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in Kd results from a reduced dissociation rate constant (from 16 s− 1 to 0.3 s− 1 in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these in vitro data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca2+]. However, for Ca2+ transients to be effective in further promoting dissociation, the elevated Ca2+ signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein–myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with in vitro data.

"The cells were immobilised on Y-shaped fibronectin patterns to give a reproducible arrangement of stress fibres spanning the apices of the cell (...) These studies confirm that for a Ca2+-dependent control of myosin filament dissociation via a calcium-binding protein such as S100A4, the mean free [Ca2+] would need to remain elevated for tens to hundreds of seconds. However, even at resting levels of Ca2+, S100A4 expressed at micromolar levels in the cytoplasm should be effective in dissociating myosin filaments."

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025356/

Barthélémy F, Kergourlay V, Lévy N, Krahn M, Bartoli M. T.P.27 Characterization of the modular domains of dysferlin for gene transfer. Neuromuscular Disorders. 2012;22(9-10):860–861.

Abstract: Dysferlin, a transmembrane protein involved in muscle membrane repair and T-tubule homeostasis is composed by several domains including seven C2 domains. Supported by the identification of specific protein partners for some of these domains, independent function of each domain has been proposed without a clear demonstration until now. Since the identification of a partially functional, naturally occurring “mini-dysferlin” protein composed of the last two C2 and C-terminal trans-membrane domains (Krahn et al., 2010), we decided to better characterize the modular properties of dysferlin. In order to test several combinations of dysferlin domains to obtain the most functional construct we employed a “mini-gene” transfer approach based on AAV vectors. Features of the native dysferlin and notably topological and phylogenic studies were conserved in the design of six different “midi-dysferlins”. We first analyzed the expression and stability of our constructs in cellular models and we found that, even if proteins show different levels of expression, their stability are not affected. Then, using micro-patterned support of culture (CYTOO technology), we showed that all our constructs are correctly addressed to the plasma membrane. This demonstrated that the full enchainment of C2 domains is not mandatory to address dysferlin to the plasma membrane. Currently, we investigate the interaction properties of these “midi-dysferlin” constructs with some of the known partners of dysferlin. Finally, based on biochemical and biological studies of these constructs expressed in mouse (C2C12) or Human (HEK) cells, we aim to confirm the functional modularity of dysferlin. Altogether, this approach will improve our knowledge of dysferlin functions and allow us to develop a mini-gene transfer therapeutic strategy.

"Using micro-patterned support of culture (...), we showed that all our constructs are correctly addressed to the plasma membrane. (...) This approach will improve our knowledge of dysferlin functions and allow us to develop a mini-gene transfer therapeutic strategy.

http://www.nmd-journal.com/article/S0960-8966%2812%2900374-4/abstract

Rosa-Ferreira C, Munro S. Arl8 and SKIP act together to link lysosomes to kinesin-1. Dev. Cell. 2011;21(6):1171–1178.

Mechano-transduction and other cell function

Vannier C, Pesty A, San-Roman MJ, Schmidt AA. The Bin/Amphiphysin/Rvs (BAR) Domain Protein Endophilin B2 Interacts with Plectin and Controls Perinuclear Cytoskeletal Architecture. J Biol Chem. 2013;288(38):27619–27637.

Abstract: Lysosomes move bidirectionally on microtubules, and this motility can be stimulated by overexpression of the small GTPase Arl8. By using affinity chromatography, we find that Arl8-GTP binds to the soluble protein SKIP (SifA and kinesin-interacting protein, aka PLEKHM2). SKIP was originally identified as a target of the Salmonella effector protein SifA and found to bind the light chain of kinesin-1 to activate the motor on the bacteria's replicative vacuole. We show that in uninfected cells both Arl8 and SKIP are required for lysosomes to distribute away from the microtubule-organizing center. We identify two kinesin light chain binding motifs in SKIP that are required for lysosomes to accumulate kinesin-1 and redistribute to the cell periphery. Thus, Arl8 binding to SKIP provides a link from lysosomal membranes to plus-end-directed motility. A splice variant of SKIP that lacks a light chain binding motif does not stimulate movement, suggesting fine-tuning by alternative splicing.

Abstract: Proteins of the Bin/amphiphysin/Rvs (BAR) domain superfamily are essential in controlling the shape and dynamics of intracellular membranes. Here, we present evidence for the unconventional function of a member of the endophilin family of BAR and Src homology 3 domain-containing proteins, namely endophilin B2, in the perinuclear organization of intermediate filaments. Using mass spectrometry analysis based on capturing endophilin B2 partners in in situ pre-established complexes in cells, we unravel the interaction of endophilin B2 with plectin 1, a variant of the cytoskeleton linker protein plectin as well as with vimentin. Endophilin B2 directly binds the N-terminal region of plectin 1 via Src homology 3-mediated interaction and vimentin indirectly via plectin-mediated interaction. The relevance of these interactions is strengthened by the selective and drastic reorganization of vimentin around nuclei upon overexpression of endophilin B2 and by the extensive colocalization of both proteins in a meshwork of perinuclear filamentous structures. By generating mutants of the endophilin B2 BAR domain, we show that this phenotype requires the BAR-mediated membrane binding activity of endophilin B2. Plectin 1 or endophilin B2 knockdown using RNA interference disturbed the perinuclear organization of vimentin. Altogether, these data suggest that the endophilin B2-plectin 1 complex functions as a membrane-anchoring device organizing and stabilizing the perinuclear network of vimentin filaments. Finally, we present evidence for the involvement of endophilin B2 and plectin 1 in nuclear positioning in individual cells. This points to the potential importance of the endophilin B2-plectin complex in the biological functions depending on nuclear migration and positioning.

Cell architecture

"Nonetheless it is clear that Arl8 and SKIP constitute a core link between lysosomal membranes and kinesin-1. This link appears to be required for maintaining the intracellular distribution of lysosomes, and for the outward movement of lysosomes in response to cytosolic acidification. This pH control of lysosome motility has been proposed to be important for cellular behavior in acidic microenvironments and, in particular, to contribute to metastasis and tumor invasion due to release of lysosomal proteases. Identification of Arl8 regulators could thus provide a route to pharmacological control of these processes."

http://www.cell.com/developmental-cell/retrieve/pii/S1534580711004540

"The nucleus position of siRNA-transfected Hela cells was determined after normalization of cell morphology on adhesive micropatterns arrays. To this end cells were plated three days after transfection at 25000 cells/cm2 in 22-mm wells onto CYTOO Chips™ Mini FN650 (Cytoo S.A., Grenoble, France) supporting H-shaped, fibronectin-coated patterns."

http://www.jbc.org/content/288/38/27619.abstract

Vignaud T, Galland R, Tseng Q, Blanchoin L, Colombelli J, Théry M. Reprogramming cell shape with laser nano-patterning. J. Cell. Sci. 2012;125(9):2134–2140.

Lombardi ML, Jaalouk DE, Shanahan CM, Burke B, Roux KJ, Lammerding J. The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton. J. Biol. Chem. 2011;286(30):26743–26753.

Abstract: Cell shape in vitro can be directed by geometrically defined micropatterned adhesion substrates. However conventional methods are limited by the fixed micropattern design, which cannot recapitulate the dynamic changes of the cell microenvironment. Here, we manipulate the shape of living cells in real time by using a tightly focused pulsed laser to introduce additional geometrically defined adhesion sites. The sub-micrometer resolution of the laser patterning allowed us to identify the critical distances between cell adhesion sites required for cell shape extension and contraction. This easy-to-handle method allows the precise control of specific actin-based structures that regulate cell architecture. Actin filament bundles or branched meshworks were induced, displaced or removed in response to specific dynamic modifications of the cell adhesion pattern. Isotropic branched actin meshworks could be forced to assemble new stress fibers locally and polarised in response to specific geometrical cues.

What was explored on CYTOO's micropatterns?...

Abstract: Maintaining physical connections between the nucleus and the cytoskeleton is important for many cellular processes that require coordinated movement and positioning of the nucleus. Nucleo-cytoskeletal coupling is also necessary to transmit extracellular mechanical stimuli across the cytoskeleton to the nucleus, where they may initiate mechanotransduction events. The LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, formed by the interaction of nesprins and SUN proteins at the nuclear envelope, can bind to nuclear and cytoskeletal elements; however, its functional importance in transmitting intracellular forces has never been directly tested. This question is particularly relevant since recent findings have linked nesprin mutations to muscular dystrophy and dilated cardiomyopathy. Using biophysical assays to assess intracellular force transmission and associated cellular functions, we identified the LINC complex as a critical component for nucleo-cytoskeletal force transmission. Disruption of the LINC complex caused impaired propagation of intracellular forces and disturbed organization of the perinuclear actin and intermediate filament networks. Although mechanically induced activation of mechanosensitive genes was normal (suggesting that nuclear deformation is not required for mechanotransduction signaling) cells exhibited other severe functional defects after LINC complex disruption; nuclear positioning and cell polarization were impaired in migrating cells and in cells plated on micropatterned substrates, and cell migration speed and persistence time were significantly reduced. Taken together, our findings suggest that the LINC complex is critical for nucleo-cytoskeletal force transmission and that LINC complex disruption can result in defects in cellular structure and function that may contribute to the development of muscular dystrophies and cardiomyopathies.

"We have demonstrated that this new and simple method for surface nano-patterning in live cell culture offers a precise control in real time of cell shape modifications and of intracellular architecture. This method should pave the way for further investigations of dynamic cellular responses to nano- and microenvironment. It also opens new possibilities to adapt ‘on the fly’ the design of new geometrical constraints to the observed cell behavior. Therefore, it will enable the fabrication of micropatterned regions during the growth of multi-cellular colonies. This will enable new insights into tissue engineering."

http://jcs.biologists.org/content/125/9/2134.long

"Using custom-developed biophysical assays, we demonstrate that disruption of the LINC complex results in defects in nuclear positioning and centrosome orientation, disturbed perinuclear organization of the actin and vimentin cytoskeleton, impaired cell motility, and reduced propagation of intracellular deformations in cells subjected to internal or external mechanical stress. Our studies offer a mechanistic explanation for these cellular defects by identifying the LINC complex as a critical element for intracellular force transmission between the cytoskeleton and the nucleus in cells, which is highly relevant to nesprin-linked diseases that primarily affect mechanically active tissues such as muscle."

http://www.jbc.org/content/286/30/26743.long

Kresh JY, Chopra A. Intercellular and extracellular mechanotransduction in cardiac myocytes. Pflugers Arch. 2011;462(1):75–87.

Abstract: Adult cardiomyocytes are terminally differentiated with minimal replicative capacity. Therefore, long-term preservation or enhancement of cardiac function depends on structural adaptation. Myocytes interact with the extracellular matrix, fibroblasts, and vascular cells and with each other (end to end; side to side). We review the current understanding of the mechanical determinants and environmental sensing systems that modulate and regulate myocyte molecular machinery and its structural organization. We feature the design and application of engineered cellular microenvironments to demonstrate the ability of cardiac cells to remodel their cytoskeletal organization and shape, including sarcomere/myofibrillar architectural topography. Cell shape-dependent functions result from complex mechanical interactions between the cytoskeleton architecture and external conditions, be they cell–cell or cell–extracellular matrix (ECM) adhesion contact-mediated. This mechanobiological perspective forms the basis for viewing the cardiomyocyte as a mechanostructural anisotropic continuum, exhibiting constant mechanosensory-driven self-regulated adjustment of the cytoskeleton through tight interplay between its force generation activity and concurrent cytoarchitectural remodeling. The unifying framework guiding this perspective is the observation that these emerging events and properties are initiated by and respond to cytoskeletal reorganization, regulated by cell–cell and cell–ECM adhesion and its corresponding (mutually interactive) signaling machinery. It is important for future studies to elucidate how cross talk between these mechanical signals is coordinated to control myocyte structure and function. Ultimately, understanding how the highly interactive mechanical signaling can give rise to phenotypic changes is critical for targeting the underlying pathways that contribute to cardiac remodeling associated with various forms of dilated and hypertrophic myopathies, myocardial infarction, heart failure, and reverse remodeling.

Dupont S, Morsut L, Aragona M, et al. Role of YAP/TAZ in mechanotransduction. Nature. 2011;474(7350):179–183.

Courtesy of Stefano Piccolo, University of Padua, Italy

"The architectural response of the cytoskeleton to the imposed cell adhesion boundary topography (shape, size) was originally studied in neonatal rat cardiac myocytes using fibronectin printed geometric solid micropatterns. When we presented the cytoskeleton with a variety of cell adhesion isotropic and multiaxial anisotropic geometries (O, H, Y, T) created by the printed fibronectin microshapes surrounded by the nonadhesive coated surface, we observed a uniquely characteristic and sensitive myofibrillar self-assembly response similar to that reported for stress fibers (...) The intent of this limited review is to outline the mechanical determinants of myocyte structural and functional cytoskeletal organization."

http://link.springer.com/article/10.1007%2Fs00424-011-0954-1

Abstract: Cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiation and cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.

Read the CYTOO Story "Nuclear transcription factors YAP/TAZ sense the stress"

http://www.cytoo.com/documents/CYTOOStories_YAP-TAZ_001.pdf

"Our findings indicate a fundamental role of the transcriptional regulators YAP and TAZ as downstream elements in how cells perceive their physical microenvironment (...) This identifies a new widespread transcriptional mechanism by which the mechanical properties of the ECM and cell geometry instruct cell behaviour. This may now shed light on how physical forces shape tissue morphogenesis and homeostasis, for example in tissues undergoing constant remodelling upon variation of their mechanical environment."

http://www.nature.com/nature/journal/v474/n7350/full/nature10137.html

Guenin-Macé L, Veyron-Churlet R, Thoulouze M-I, et al. Mycolactone activation of Wiskott-Aldrich syndrome proteins underpins Buruli ulcer formation. Journal of Clinical Investigation. 2013; Mar 15 [epub ahead of print].

Abstract: Mycolactone is a diffusible lipid secreted by the human pathogen Mycobacterium ulcerans, which induces the formation of open skin lesions referred to as Buruli ulcers. Here, we show that mycolactone operates by hijacking the Wiskott-Aldrich syndrome protein (WASP) family of actin-nucleating factors. By disrupting WASP autoinhibition, mycolactone leads to uncontrolled activation of ARP2/3-mediated assembly of actin in the cytoplasm. In epithelial cells, mycolactone-induced stimulation of ARP2/3 concentrated in the perinuclear region, resulting in defective cell adhesion and directional migration. In vivo injection of mycolactone into mouse ears consistently altered the junctional organization and stratification of keratinocytes, leading to epidermal thinning, followed by rupture. This degradation process was efficiently suppressed by coadministration of the N-WASP inhibitor wiskostatin. These results elucidate the molecular basis of mycolactone activity and provide a mechanism for Buruli ulcer pathogenesis. Our findings should allow for the rationale design of competitive inhibitors of mycolactone binding to N-WASP, with anti–Buruli ulcer therapeutic potential.

Nabavi N, Pustylnik S, Harrison RE. Rab GTPase Mediated Procollagen Trafficking in Ascorbic Acid Stimulated Osteoblasts. PLoS ONE. 2012;7(9):e46265.

Abstract: Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly up-regulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone protein secretion and deposition. Protein trafficking along the exocytic and endocytic pathways is aided by many molecules, with Rab GTPases being master regulators of vesicle targeting. In this study, we used microarray analysis to identify the Rab GTPases that are up-regulated during a 5-day AA differentiation of OBs, namely Rab1, Rab3d, and Rab27b. Further, we investigated the role of identified Rabs in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane utilizing Rab dominant negative (DN) expression. We also observed that experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs partially alleviated this negative feedback mechanism and resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels was also observed implicating the functional importance of Rab1, Rab3d and Rab27b in these major collagen-producing cells.

"To analyze the effect of mycolactone on the cytoskeleton, we selected the HeLa cell line, as a model of anchorage-dependent cells of the human epithelial system, as a model of anchorage-dependent cells of the human epithelial system, and the Jurkat cell line of human T lymphocytes, to reflect the activity of mycolactone on nonadherent, immune cells (...) HeLa cells were plated on Y-shaped, fibronectin-coated micropatterns"

http://www.jci.org/articles/view/66576

Planagumà J, Minsaas L, Pons M, Myhren L, Garrido G, Aragay AM. Filamin A-Hinge Region 1-EGFP: A Novel Tool for Tracking the Cellular Functions of Filamin A in Real-Time. PLoS ONE. 2012;7(8):e40864.

"An interesting extracellular collagen phenotype pattern observed in mutant cells plated on CYTOO slides was the disappearance of long collagen fibrils extending and connecting differentiating cells. Normally, confluent differentiating OBs on glass coverslips secrete characteristic extensive collagen fibrils and their absence in spatially separated cells may suggest a mechanistic link between cell-cell contact sites and proper collagen fibrillogenesis."

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0046265

Cell adhesion

Abstract:

Background

Filamin A (FLNa) is an actin-crosslinking protein necessary for stabilizing the cell surface, organizing protrusive activity and for promoting efficient cellular translocation. Recently, our group demonstrated the requirement of FLNa for the internalization of the chemokine receptor CCR2B.

Methodology and principal findings

In order to study the role of FLNa in vitro and in real-time, we have developed a fluorescent FLNa-EGFP construct. In this novel imaging tool, we introduced the EGFP-tag inside the flexible hinge 1 region of FLNa between two calpain cleavage sites. Our findings indicate that the FLNa-EGFP construct was correctly expressed, cleaved by calpain and colocalized with actin filaments as shown by immunostaining experiments in the human melanoma cell lines A7 (FLNa-repleted) and M2 (FLNa-deficient). In addition, scanning-electron microscopy (SEM) and micropatterning studies also provided clear evidence that the cell rigidity was restored. FLNa-EGFP allowed us to demonstrate the interaction of FLNa with the chemokine receptor CCR2B in endocytic vesicles after CCL2 ligand stimulation. Through live-cell imaging studies we show that the CCR2B receptor in Rab5-positive vesicles moves along filamin A-positive fibers.

Significance

Taken together, these results outline the functionality of the FLNa-EGFP and the importance of filamin A for receptor internalization and movement into endocytic vesicles.

Mitochondrial networks

Chevrollier A, Cassereau J, Ferré M, et al. Standardized mitochondrial analysis gives new insights into mitochondrial dynamics and OPA1 function. Int. J. Biochem. Cell Biol. 2012;44(6):980–988.

"These findings open up new opportunities to use this tool for investigating and characterizing the potential interaction of FLNa with chemokine receptors and its role in the vesicular trafficking."

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0040864

Abstract: Mitochondria form dynamic tubular networks through processes of fission and fusion. Defect in mitochondrial dynamics lead to various pathologies, including several common and some rare neurodegenerative disorders. OPA1 and MFN2 are two key players in mitochondrial fusion associated with Autosomal Dominant Optic Atrophy and Charcot Marie Tooth neuropathy type 2A respectively. We used micropatterned coverslips to standardize the visualization of mitochondrial distribution in skin fibroblasts. In fibroblasts from affected patients, mutations in the OPA1 and MFN2 genes were found to affect the volume and cellular distribution of mitochondria. In G1/S cell cycle phase, mitochondria emerging from the microtubule organizing centre may be crucial to mitochondrial biogenesis since it appeared to be protected against mitochondrial fragmentation induced by OPA1 mutations. The standardized quantitative analysis of the mitochondrial network and the description of mitochondrial subcellular distribution should lead to better diagnostic criteria for mitochondrial diseases and yield new insights into mitochondrial dysfunction in disease and aging

Nascimento E, Guzman-Quevedo O, Delacourt N, et al. Long-Lasting Effect of Perinatal Exposure to L-tryptophan on Circadian Clock of Primary Cell Lines Established from Male Offspring Born from Mothers Fed on Dietary Protein Restriction. PLoS ONE. 2013;8(2):e56231.

"The potential application of the methods proposed are illustrated here by the study of mitochondria affected by pathogenic mutations of the OPA1 and MFN2 genes, which are known to be involved in mitochondrial fusion. The standardized quantitative analysis of the mitochondrial network and the description of mitochondrial subcellular distribution should lead to the establishment of better diagnostic criteria for mitochondrial diseases and yield new insights into these disorders."

http://www.sciencedirect.com/science/article/pii/S1357272512000970

Abstract

Background & Aims

Maternal undernutrition programs metabolic adaptations which are ultimately detrimental to adult. L-tryptophan supplementation was given to manipulate the long-term sequelae of early-life programming by undernutrition and explore whether cultured cells retain circadian clock dysregulation.

Methods

Male rat pups from mothers fed on low protein (8%, LP) or control (18%, CP) diet were given, one hour before light off, an oral bolus of L-tryptophan (125 mg/kg) between Day-12 and Day-21 of age. Body weight, food intake, blood glucose along with the capacity of colonization of primary cells from biopsies were measured during the young (45–55 days) and adult (110–130 days) phases. Circadian clock oscillations were re-induced by a serum shock over 30 hours on near-confluent cell monolayers to follow PERIOD1 and CLOCK proteins by Fluorescent Linked ImmunoSorbent Assay (FLISA) and period1 and bmal1 mRNA by RT-PCR. Cell survival in amino acid-free conditions were used to measure circadian expression of MAP-LC3B, MAP-LC3B-FP and Survivin.

Results

Tryptophan supplementation did not alter body weight gain nor feeding pattern. By three-way ANOVA of blood glucose, sampling time was found significant during all phases. A significant interaction between daily bolus (Tryptophan, saline) and diets (LP, CP) were found during young (p = 0.0291) and adult (p = 0.0285) phases. In adult phase, the capacity of colonization at seeding of primary cells was twice lower for LP rats. By three-way ANOVA of PERIOD1 perinuclear/nuclear immunoreactivity during young phase, we found a significant effect of diets (p = 0.049), daily bolus (p<0.0001) and synchronizer hours (p = 0.0002). All factors were significantly interacting (p = 0.0148). MAP-LC3B, MAP-LC3B-FP and Survivin were altered according to diets in young phase.

Conclusions

Sequelae of early-life undernutrition and the effects of L-tryptophan supplementation can be monitored non-invasively by circadian sampling of blood D-glucose and on the expression of PERIOD1 protein in established primary cell lines.

"To test the homogeneity of cellular preparation and the capacity to adhere to a substratum, we have seeded established cell lines of LP and CP groups onto Cytoo chamber starter kit. The Cytoo kit proposes different preset forms onto plastic substratum pre-coated with fibronectin (...) data indicate that rats derived from LP dams were less prone to give rise to primary cell cultures after the young phase, irrespectively of a daily bolus of L-tryptophan".

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0056231

Pelikan A, Sillibourne J, Miserey-Lenkei S, Carlier-Grynkorn F, Goud B, Tran PT. Chapter 7 - Studying Mitochondria and Microtubule Localization and Dynamics in Standardized Cell Shapes. In: John J. Correia and Leslie Wilson, eds. Methods in Cell Biology. Vol 115. Academic Press; 2013:97–108.

Schiller HB, Hermann M-R, Polleux J, et al. Beta1- and Alphav-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments. Nat Cell Biol. 2013; May 26 [epub ahead of print].

© Plos One 2013 Nascimento et al.

Courtesy of the lab of Drs Donna McPhie and Bruce Cohen

Harvard Medical School, MA, USA

Abstract: Mammalian cells show a large diversity in shape and are both shape-changing and mobile when cultured on conventional uniform substrates. The use of micropatterning techniques limits the number of variable parameters, by imposing shape and standardized adhesive areas on the cells, which facilitates analysis. By changing size or shape of the micropattern, for example, forcing a polar axis on the cell, it is possible to study how these parameters impact organelle organization, distribution, and dynamics inside the cell. To study the mitochondrial network, which is composed of dynamic tubular organelles dependent on the microtubule cytoskeleton for its distribution, it is important to be able to distinguish between distinct mitochondria. Here, we present a practical method with which we spread the cells on large patterns created with deep UV technique, which not only makes the cells uniform in size and shape as well as immobile, and therefore easier to compare and analyze, but also expands the mitochondrial network and allows for an easier tracking of appropriately labeled individual mitochondria.

Abstract: How different integrins that bind to the same type of extracellular matrix protein mediate specific functions is unclear. We report the functional analysis of beta1- and alpha v-class integrins expressed in pan-integrin-null fibroblasts seeded on fibronectin. Reconstitution with beta1-class integrins promotes myosin-II-independent formation of small peripheral adhesions and cell protrusions, whereas expression of alpha v-class integrins induces the formation of large focal adhesions. Co-expression of both integrin classes leads to full myosin activation and traction-force development on stiff fibronectin-coated substrates, with alpha v-class integrins accumulating in adhesion areas exposed to high traction forces. Quantitative proteomics linked v-class integrins to a GEF-H1-RhoA pathway coupled to the formin mDia1 but not myosin II, and alpha5beta1 integrins to a RhoA-Rock-myosin II pathway. Our study assigns specific functions to distinct fibronectin-binding integrins, demonstrating that alpha5beta1 integrins accomplish force generation, whereas alpha v-class integrins mediate the structural adaptations to forces, which cooperatively enable cells to sense the rigidity of fibronectin-based microenvironments.

Read the CYTOO Story "Mitochondria dynamics in focus"

http://www.cytoo.com/documents/Cytoo-Stories-Mitochondria.pdf

"Micropatterning technique is robust. A comparison between RPE1 cells seeded onto nonpatterned surface and cells seeded onto rectangular patterned surface. Nonpatterned cells can adopt many shapes, making analysis of dynamics subcellular structures such as mitochondria and microtubules challenging. Bar 10 mm. Patterned shapes are repeatable, given rise to standardized cell shapes and size, making analysis of dynamics subcellular structures such as mitochondria and microtubules less challenging."

http://www.sciencedirect.com/science/article/pii/B9780124077577000074

"Adhesion maturation and trailing edge retraction in migrating fibroblasts requires coordinated control of myosin-II-mediated cell contractility. We measured myosin II activity using fibronectincoated X- or crossbow-shaped micropatterns, which report subtle changes in myosin II activity and traction forces along nonadhesive edges."

http://www.nature.com/ncb/journal/vaop/ncurrent/full/ncb2747.html

Rodríguez-Fraticelli AE, Martín-Belmonte F. Mechanical control of epithelial lumen formation. Small GTPases. 2013;4(2).

Abstract: Epithelial cells differentiate and polarize to build complete epithelial organs during development. The study of epithelial morphogenesis is instrumental to the understanding of disease processes where epithelial polarity is disrupted. Recently, we demonstrated that matrix-induced cell confinement controls the acquisition of three-dimensional epithelial polarity, by modulating the initiation of the apical membrane to form a central lumen (J Cell Biol 2012; 198:1011-1026). Cell confinement can be achieved by use of micropatterned culture chips that allow precise micrometric-scale control of the cell adhesion surface and its composition. Using micropattern chips, we demonstrated that polarizing epithelial cells require high confinement conditions to properly position the centrosome and the trafficking machinery toward the cell-cell contacts and to initiate lumen morphogenesis. Low confinement induces LKB1 and RhoA-mediated cell contractility, which inhibits this mechanism for lumen formation. Deactivation of Myosin-II-mediated contractility rescued normal lumen initiation in low confinement conditions. Our results indicate that a mechanotransduction pathway coordinates nuclear and centrosome positioning to initiate epithelial morphogenesis. Here we discuss the potential candidates that control this process, specifically the polarized activation of Rho and Rab-family GTPases, and also a group of recently characterized nuclear transcription factors.

"These results indicate that cell contractility and primary cilia formation are mutually exclusive processes in cellular physiology. Furthermore, they introduce the possibility that other processes required for epithelial morphogenesis could be modulated by contractility and thus, more easily studied using micropatterns."

http://www.landesbioscience.com/journals/smallgtpases/article/24303/?show_full_text=true&

Woldemichael GM, Turbyville TJ, Linehan WM, McMahon JB. Carminomycin I is an apoptosis inducer that targets the Golgi complex in clear cell renal carcinoma cells. Cancer Res. 2011;71(1):134–142.

Cancer research

Rodríguez-Fraticelli AE, Auzan M, Alonso MA, Bornens M, Martín-Belmonte F. Cell confinement controls centrosome positioning and lumen initiation during epithelial morphogenesis. J Cell Biol. 2012. 198(6):1011-23.

Abstract: Clear cell renal cell carcinoma (CCRCC) evolves due to mutations in the Von Hippel-Lindau (VHL) tumor suppressor gene. Although the loss of VHL enables survival and proliferation of CCRCC cells, it is also expected to introduce vulnerabilities that may be exploited for therapeutics discovery. To this end, we developed a high-throughput screen to identify small molecules derived from plants, microorganisms, and marine organisms to which CCRCC cells are sensitive. Screening over 8,000 compounds using this approach, we report here the identification of the microbially derived compound carminomycin I (CA) as an effective inhibitor of VHL-defective (VHL(-/-)) CCRCC cell proliferation. CA also induced apoptosis in CCRCC cells by a mechanism independent of p53 or hypoxia-inducible factor 2. We found that P-glycoprotein (P-gp) sequestered CA within the Golgi complex. Interestingly, Golgi sequestration was critical for the antiproliferative effects of CA and P-gp inhibitors abrogated this activity. Furthermore, CA induced cleavage of the Golgi protein p115 and the translocation of its C-terminal fragment to the nucleus. Finally, examination of the activity of the VHL-interacting Golgi protein, endoplasmic reticulum-Golgi intermediate compartment, ERGIC-53 showed that VHL could mediate protection from CA in CCRCC cells. Our natural product-based screening approach has revealed the P-gp-mediated localization of anticancer compounds within the Golgi in CCRCC cells as a potential strategy of targeting VHL-deficient CCRCC cells.

Abstract: Epithelial organ morphogenesis involves sequential acquisition of apicobasal polarity by epithelial cells and development of a functional lumen. In vivo, cells perceive signals from components of the extracellular matrix (ECM), such as laminin and collagens, as well as sense physical conditions, such as matrix stiffness and cell confinement. Alteration of the mechanical properties of the ECM has been shown to promote cell migration and invasion in cancer cells, but the effects on epithelial morphogenesis have not been characterized. We analyzed the effects of cell confinement on lumen morphogenesis using a novel, micropatterned, three-dimensional (3D) Madin-Darby canine kidney cell culture method. We show that cell confinement, by controlling cell spreading, limits peripheral actin contractility and promotes centrosome positioning and lumen initiation after the first cell division. In addition, peripheral actin contractility is mediated by master kinase Par-4/LKB1 via the RhoA-Rho kinase-myosin II pathway, and inhibition of this pathway restores lumen initiation in minimally confined cells. We conclude that cell confinement controls nuclear-centrosomal orientation and lumen initiation during 3D epithelial morphogenesis.

Morphogenesis

"Cells grown on O- and L-shaped fibronectin micropatterns on glass chips to normalize cell position, shape, polarity, and internal organelle positioning also confirmed the presence of P-gp in the Golgi in addition to the cell membrane (...) our findings illustrate a potential novel approach for targeting VHL/ clear cell renal carcinoma. Our findings also underscore the utility of natural products in yielding compounds that may be used either in therapeutic development or as bioprobes."

http://cancerres.aacrjournals.org/content/71/1/134.long

"In the present study, we characterized the role of cell confinement in lumen formation using a new methodology to analyze 3D epithelial morphogenesis in micropatterns. We found that cell confinement, which modifies the actomyosin II–mediated contractility, is able to regulate epithelial polarity and lumen formation and the positioning of the centrosome and the nucleus. In conditions of low confinement, cell spreading increases peripheral actin contractility, which in turn impairs the initiation of lumen formation. Peripheral actomyosin contractility maintains centrosome positioning at the center of the cell perimeter and forces nuclear positioning toward the cell–cell junctions".

http://jcb.rupress.org/content/198/6/1011.abstract

Aragona M, Panciera T, Manfrin A, et al. A Mechanical Checkpoint Controls Multicellular Growth through YAP/TAZ Regulation by Actin-Processing Factors. Cell. 2013; Aug 15 [epub ahead of print].

Key cellular decisions, such as proliferation or growth arrest, typically occur at spatially defined locations within tissues. Loss of this spatial control is a hallmark of many diseases, including cancer. Yet, how these patterns are established is incompletely understood. Here, we report that physical and architectural features of a multicellular sheet inform cells about their proliferative capacity through mechanical regulation of YAP and TAZ, known mediators of Hippo signaling and organ growth. YAP/TAZ activity is confined to cells exposed to mechanical stresses, such as stretching, location at edges/curvatures contouring an epithelial sheet, or stiffness of the surrounding extracellular matrix. We identify the F-actin-capping/severing proteins Cofilin, CapZ, and Gelsolin as essential gatekeepers that limit YAP/TAZ activity in cells experiencing low mechanical stresses, including contact inhibition of proliferation. We propose that mechanical forces are overarching regulators of YAP/TAZ in multicellular contexts, setting responsiveness to Hippo, WNT, and GPCR signaling.

Woldemichael GM, Turbyville TJ, Vasselli JR, Linehan WM, McMahon JB. Lack of a Functional VHL Gene Product Sensitizes Renal Cell Carcinoma Cells to the Apoptotic Effects of the Protein Synthesis Inhibitor Verrucarin A. Neoplasia. 2012;14(8):771–777.

"We employed microfabrication methods to stamp fibronectin-coated substrates of defined shape and area. This set-up allows studying how patterns of mechanical forces generate patterned growth within a monolayer: cells located at the borders of the island experience higher mechanical stress thancells located in the center."

http://www.sciencedirect.com/science/article/pii/S0092867413009513

Abstract: Verrucarin A (VA) is a small molecule derived from the fungal plant pathogen Myrothecium verrucaria and was identified as a selective inhibitor of clear cell renal cell carcinoma (CCRCC) cell proliferation in a high-throughput screen of a library of naturally occurring small molecules. CCRCC arises as a result of loss-of-function mutations in the von Hippel–Lindau (VHL) gene. Here we show that VA inhibits protein translation initiation culminating in apoptosis through the extrinsic signaling pathway. Reintroduction of the VHL gene in CCRCC cells afforded resistance to VA’s apoptotic effects. This resistance is mediated in part by the formation of stress granules that entrap signaling molecules that initiate the apoptotic signaling cascade. The VHL gene product was found to be a component of stress granules that develop as result of VA treatment. These findings reveal an important role for the VHL gene product in cytotoxic stress response and have important implications for the rational development of VA-related compounds in chemotherapeutic targeting of CCRCC.

"We carried out immunofluorescence colocalization experiments on VA-treated cells attached to Y-shaped fibronectin micropatterns on glass chips. This allowed for better quantification through normalization of cell shape and internal cell organization (...) Taken together, our results suggest that selective targeting of VHL−/− cells might be possible in CCRCC through the use of specific inhibitors of translation initiation. It seems that VHL+/+ cells are better able to handle the stress induced by such compounds. The evidence provided in this study suggests the presence of vulnerability in CCRCC cells that may be exploited for therapeutics development."

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3431183/

Verhulsel M, Vignes M, Descroix S, Malaquin L, Vignjevic DM, Viovy J-L. A review of microfabrication and hydrogel engineering for micro-organs on chips. Biomaterials. 2013; Dec 04 [epub ahead of print].

Image courtesy of Mithila Burute (CEA)

Abstract: This review highlights recent trends towards the development of in vitro multicellular systems with definite architectures, or “organs on chips”. First, the chemical composition and mechanical properties of the scaffold have to be consistent with the anatomical environment in vivo. In this perspective, the flourishing interest in hydrogels as cellular substrates has highlighted the main parameters directing cell differentiation that need to be recapitulated in artificial matrix. Another scaffold requirement is to act as a template to guide tissue morphogenesis. Therefore specific microfabrication techniques are required to spatially pattern the environment at microscale. 2D patterning is particularly efficient for organizing planar polarized cell types such as endothelial cells or neurons. However, most organs are characterized by specific sub units organized in three dimensions at the cellular level. The reproduction of such 3D patterns in vitro is necessary for cells to fully differentiate, assemble and coordinate to form a coherent micro-tissue. These physiological microstructures are often integrated in microfluidic devices whose controlled environments provide the cell culture with more life-like conditions than traditional cell culture methods. Such systems have a wide range of applications, for fundamental research, as tools to accelerate drug development and testing, and finally, for regenerative medicine.

"2D cell culture substrates integrating chemical patterns are available commercially via CYTOO Company (www.cytoo.com). This company markets a wide variety of micropatterns, each one dedicated to a specific use, from single cell analysis to multicellular networks."

http://www.sciencedirect.com/science/article/pii/S0142961213013744

Courtesy of Minerva Bosch and Alejo Rodriguez-Fraticelli from the Laboratory of Fernando Martin-Belmonte, CBMSO, Universidad Autonoma de Madrid, Spain

Read the CYTOO Story "Tumor ECM: the highway to cell"

http://www.cytoo.com/documents/CytooStories_TumorCellMigration_web3_000.pdf

Rodríguez-Fraticelli AE, Martín-Belmonte F. Chapter 7 - Methods for Analysis of Apical Lumen Trafficking Using Micropatterned 3D Systems. In: Franck Perez and David J. Stephens, ed. Methods in Cell Biology.Vol Volume 118. Methods for Analysis of Golgi Complex Function. Academic Press; 2013:105–123.

Abstract: Epithelial organs are made of interconnected branched networks of tubules, with a central lumen lined by a monolayer of epithelial cells. Certain epithelial cell lines can be converted into organotypic cultures by the addition of extracellular matrix components. When cultured in these conditions, epithelial cells reorient the axis of polarity, reorganize the membrane surfaces, and transport apical proteins to form the lumen in a process that recapitulates essential aspects of de novo apical membrane formation during epithelial organ morphogenesis. Micropatterns are a simple technique that allows cell culture in a controlled adhesive environment with extremely high precision, close to the nanometer scale. We have recently developed a method to culture MDCK cysts on micropatterns of different sizes and composition. Using this method we found that changes in micropattern shape and size can be used to modify cell contractility to understand its contribution to apical membrane formation. When imaging cysts on micropatterns the main advantage is that apical-directed vesicle trafficking is visualized in the x–y plane, which presents higher resolution on confocal microscopes. Thus, the use of micropatterns is an efficient setup to analyze polarized secretion with unprecedented higher resolution in both time and space.

"Although different methods exist for producing micropatterns for cell culture, we recommend CYTOOchips (available worldwide from www.cytoo.com). CYTOOchips present highly reproducible conditions (shape, size, coating) for each micropattern, which is an advantage for high-throughput experiments."

http://www.sciencedirect.com/science/article/pii/B9780124171640000070

Phenotypic screening

Tseng Q. Etude d’architecture multicellulaire avec le microenvironnement controlé. [dissertation]. Grenoble: Université de Grenoble; 2011.

Thesis

cell architecture, micropatterns

Stem cells

Synapse formation

Abstract: This thesis dissertation is comprised of three major parts. The first part devotes to all the technological developments that have been realized in my thesis study. These developments in microfabrication, in image acquisition and analysis, and in the traction force analysis had solved various problems we have encountered during our study of epithelial architecture. The second part describes the study of the spatial organization of the adhesion systems in epithelia. From their polarity, their functioning, to their remodeling, the epithelial architecture is deeply linked with the adhesion systems. With the capability to well define the location of cell-matrix interaction, we examined how the intercellular adhesion was organized according to the cell-matrix adhesion. Our results highlighted the instructive role of cell-matrix adhesion in organizing the intercellular adhesion. This organization subsequently governed the internal polarity which was indicated by the centrosome positioning. During epithelial remodeling, both the adhesion system and internal polarity were subjected to modification. Nevertheless they could be regulated differently depending on the context of remodeling. The last part is focused on the physical aspect of the epithelial architecture. Apart from the biochemical signaling network, mechanical force is also a substantial ingredient in morphogenesis. Together with our techniques in micropatterning the soft gel, the development of software for traction force microscope, and our knowledge of cell-cell positioning, we were able to analyze precisely the mechanical property of the multicellular architecture. We found that the cellular contractility was modulated by the spatial organization of the adhesion system. It permitted us to complete the current physical model of epithelial geometry with an anisotropic term for contractility. This new physical model could effectively account for the cell positioning on various matrix geometries.

"With a constrained cell geometry on micropattern, localization of cellular proteins by immunofluorescent labeling becomes more comparable from cell to cell. We can easily make statistical analysis on protein localization by making a virtual cell where each pixel value corresponds to the mean or standard deviations of intensities of the same pixel from all the samples. This could reveal subtile variations which are not readily perceivable on individual immunofluorescent image."

http://tel.archives-ouvertes.fr/tel-00622264/

Schauer K, Grossier J-P, Duong T, et al. A Novel Organelle Map Framework for High-Content Cell Morphology Analysis in High Throughput. J Biomol Screen. 2013; Aug 16 [epub ahead of print].

A screening procedure was developed that takes advantage of the cellular normalization by micropatterning and a novel quantitative organelle mapping approach that allows unbiased and automated cell morphology comparison using black-box statistical testing. Micropatterns of extracellular matrix proteins force cells to adopt a reproducible shape and distribution of intracellular compartments avoiding strong cell-to-cell variation that is a major limitation of classical culture conditions. To detect changes in cell morphology induced by compound treatment, fluorescently labeled intracellular structures from several tens of micropatterned cells were transformed into probabilistic density maps. Then, the similarity or difference between two given density maps was quantified using statistical testing that evaluates differences directly from the data without additional analysis or any subjective decision. The versatility of this organelle mapping approach for different magnifications and its performance for different cell shapes has been assessed. Density-based analysis detected changes in cell morphology due to compound treatment in a small-scale proof-of-principle screen demonstrating its compatibility with high-throughput screening. This novel tool for high-content and high-throughput cellular phenotyping can potentially be used for a wide range of applications from drug screening to careful characterization of cellular processes.

biotechnology, micropatterns

Kauffmann P. Lévitation diamagnétique sur micro-aimants: applications a la microfluidique digitale et à la biologie. [dissertation]. Grenoble: Institut National Polytechnique; 2009.

"Our organelle mapping framework in micropatterned cells may represent a major step toward quantitative, high-content, and fast analysis for automated detection of cell morphology changes in high-throughput screens. This framework has the potential to become a gold standard for cell phenotyping in high-troughput screening."

http://jbx.sagepub.com/content/early/2013/08/15/1087057113497399.abstract

Abstract: Diamagnetic levitation is one of the rare way to compensate action of gravity. This kind of repulsion is negligible at our scale. However, at microscale, this effect becomes significant and can achieve levitation of diamagnetic objects. Through the development of micromagnets, analytical and numerical models, and experiments, applications of diamagnetic levitation of microdroplets and trapping of cells in paramagnetic media is explored. It is shown that diamagnetic levitation allows quantifying interactions between levitating charged droplets. The behaviour of cells trapped in paramagnetic medium is also analysed. This study opens attractive alternatives to cells sorting, based on size, magnetic susceptibilities and endocytoticity. Finally, remote handling of levitating droplets is proposed and simulated by coupling diamagnetic levitation and dielectrophoresis.

Read the CYTOO Story "Quantifying the undetectable: Probabilistic density maps on normalized cells"

http://www.cytoo.com/documents/CytooStories-Organelle-Map_000.pdf

"Currently, microcontact printing is the most robust, simple and easy method. Microcontact printing techniques have been refined and simplified, and enabled single cells adhesion on micropatterns. Nowadays, this technique is being used by a CEA start-up, the CYTOO company. These micropatterns enable positioning cells in a perfectly reproducible manner, which simplifies analysis. Micropatterns offer not only a cell matrix, but impose also a specific cell shape, and then intracellular architecture and cell polarity." (translated by CYTOO)

http://tel.archives-ouvertes.fr/tel-00455369/

Courtesy of Sravanti Kusuma

Department of Biomedical Engineering

Johns Hopkins University

Courtesy of Anselme Perrier, I-Stem

Degot S, Auzan M, Chapuis V, et al. Improved visualization and quantitative analysis of drug effects using micropatterned cells. J Vis Exp. 2010;(46).

"L’impression par microcontact est actuellement la méthode la plus robuste, simple et rapide. Les techniques d’impression par microcontact se sont affinées et simplifiées et ont permis l’adhésion de cellules uniques sur des micromotifs. A l’heure actuelle, cette technique est utilisée par une start-up du CEA, la société Cytoo. Ces micromotifs permettent de positionner des cellules de manière parfaitement reproductible ce qui rend l’analyse plus facile. En plus d’offrir un matriçage des cellules, les micromotifs imposent aussi la forme des cellules et donc l’organisation intracellulaire et la polarité de la cellule"

Abstract: To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck (1-2). To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

"Micropatterns facilitate the visualization and quantification of drug effects, especially at low concentrations. The replacement of homogenous planar substrates with adhesive micropatterns for cell-based assays improves the accuracy, sensitivity and quality of data produced. In consequence, fewer cells are needed for analysis in order to achieve robust statistical outcomes 3. Adhesive micropatterns offer a real opportunity for improving functional studies and exploring phenotypic changes at lower drug concentrations in order to avoid inducing toxic or indirect drug effects. If adequate screening platforms are used, micropatterns can meet the demands of pharmaceutical companies for improving gene/drug screening applications."

http://www.jove.com/video/2514/improved-visualization-quantitative-analysis-drug-effects-using?ID=2514

Czöndör K, Garcia M, Argento A, et al. Micropatterned substrates coated with neuronal adhesion molecules for high-content study of synapse formation. Nat Commun. 2013;4(2252).

Pryzhkova MV, Harris GM, Ma S, Jabbarzadeh E. Patterning Pluripotent Stem Cells at a Single Cell Level. J Biomater Tissue Eng. 2013;3(4):461–471

Schauer K, Duong T, Bleakley K, Bardin S, Bornens M, Goud B. Probabilistic density maps to study global endomembrane organization. Nat. Methods. 2010;7(7):560–566.

Freida D, Lecourt S, Cras A, et al. Human Bone Marrow Mesenchymal Stem Cells Regulate Biased DNA Segregation in Response to Cell Adhesion Asymmetry. Cell Rep. 2013;Oct 17 [epub ahead of print].

Duong T, Goud B, Schauer K. Closed-form density-based framework for automatic detection of cellular morphology changes. Proc. Natl. Acad. Sci. U.S.A. 2012;109(22):8382–8387.

Abstract: We developed a computational imaging approach that describes the three-dimensional spatial organization of endomembranes from micromanipulation-normalized mammalian cells with probabilistic density maps. Applied to several well-known marker proteins, this approach revealed the average steady-state organization of early endosomes, multivesicular bodies or lysosomes, endoplasmic reticulum exit sites, the Golgi apparatus and Golgi-derived transport carriers in crossbow-shaped cells. The steady-state organization of each tested endomembranous population was well-defined, unique and in some cases depended on the cellular adhesion geometry. Density maps of all endomembrane populations became stable when pooling several tens of cells only and were reproducible in independent experiments, allowing construction of a standardized cell model. We detected subtle changes in steady-state organization induced by disruption of the cellular cytoskeleton, with statistical significance observed for just 20 cells. Thus, combining micropatterning with construction of endomembrane density maps allows the systematic study of intracellular trafficking determinants.

Abstract: A primary method for studying cellular function is to examine cell morphology after a given manipulation. Fluorescent markers attached to proteins/intracellular structures of interest in conjunction with 3D fluorescent microscopy are frequently exploited for functional analysis. Despite the central role of morphology comparisons in cell biological approaches, few statistical tools are available that allow biological scientists without a high level of statistical training to quantify the similarity or difference of fluorescent images containing multifactorial information. We transform intracellular structures into kernels and develop a multivariate two-sample test that is nonparametric and asymptotically normal to directly and quantitatively compare cellular morphologies. The asymptotic normality bypasses the computationally intensive calculations used by the usual resampling techniques to compute the P-value. Because all parameters required for the statistical test are estimated directly from the data, it does not require any subjective decisions. Thus, we provide a black-box method for unbiased, automated comparison of cell morphology. We validate the performance of our test statistic for finite synthetic samples and experimental data. Employing our test for the comparison of the morphology of intracellular multivesicular bodies, we detect changes in their distribution after disruption of the cellular microtubule cytoskeleton with high statistical significance in fixed samples and live cell analysis. These results demonstrate that density-based comparison of multivariate image information is a powerful tool for automated detection of cell morphology changes. Moreover, the underlying mathematics of our test statistic is a general technique, which can be applied in situations where two data samples are compared.

Biased DNA segregation is a mitotic event in which the chromatids carrying the original template DNA strands and those carrying the template copies are not segregated randomly into the two daughter cells. Biased segregation has been observed in several cell types, but not in human mesenchymal stem cells (hMSCs), and the factors affecting this bias have yet to be identified. Here, we have investigated cell adhesion geometries as a potential parameter by plating hMSCs from healthy donors on fibronectin-coated micropatterns. On symmetric micropatterns, the segregation of sister chromatids to the daughter cells appeared random. In contrast, on asymmetric micropatterns, the segregation was biased. This sensitivity to asymmetric extracellular cues was reproducible in cells from all donors but was not observed in human skin-derived fibroblasts or in a fibroblastic cell line used as controls. We conclude that the asymmetry of cell adhesion is a major factor in the regulation of biased DNA segregation in hMSCs.

Studying the roles of different proteins and the mechanisms involved in synaptogenesis is hindered by the complexity and heterogeneity of synapse types, and by the spatial and temporal unpredictability of spontaneous synapse formation. Here we demonstrate a robust and high-content method to induce selectively presynaptic or postsynaptic structures at controlled locations. Neurons are cultured on micropatterned substrates comprising arrays of micron-scale dots coated with various synaptogenic adhesion molecules. When plated on neurexin-1beta-coated micropatterns, neurons expressing neuroligin-1 exhibit specific dendritic organization and selective recruitment of the postsynaptic scaffolding molecule PSD-95. Furthermore, functional AMPA receptors are trapped at neurexin-1beta dots, as revealed by live-imaging experiments. In contrast, neurons plated on SynCAM1-coated substrates exhibit strongly patterned axons and selectively assemble functional presynapses. N-cadherin coating, however, is not able to elicit synapses, indicating the specificity of our system. This method opens the way to both fundamental and therapeutic studies of various synaptic systems.

Studies of cell-extracellular matrix (ECM) interactions at a single cell level have drawn interest from scientists around the world. Subcellular ECM micropatterning techniques allow researchers to control cell shape, migration, and spindle orientation during mitosis potentially influencing the stem cell fate. Generally these studies have been limited to somatic cells rather than human pluripotent stem cells (hPSCs) which are capable of enormous differentiation potential. hPSCs require a defined ECM for attachment and express characteristic integrins mediating cell-substrate interactions. hPSCs also rely on cell–cell contacts for survival and to maintain self-renewal properties, but these circumstances also significantly limit hPSC observation at a single cell level. In addition, currently available methods for ECM micropatterning generally require a facility with trained personnel and intricate equipment to produce protein micropatterns. To overcome this problem, we have developed a protocol for vitronectin micropatterning using simple UV/ozone modification of polystyrene. Single hPSCs were able to attach and form characteristic stress fibers and focal adhesions similar to somatic cell types which demonstrate hPSC responsiveness to extracellular adhesive cues. Micropatterned hPSCs were able to be cultured for up to 48 hours while maintaining expression of pluripotency-associated transcription factor OCT4. Although further studies are necessary, the results of our investigation will potentially have a large impact on cell regenerative medicine and tissue engineering.

"To avoid the limitations of classical approaches that use unconstrained, dynamic cell shapes, we used microfabricated patterns, which enforce cells to take a certain shape and prevent their migration14. Micropatterns allow for control of organelle organization, and we ascertained a normal cell cycle and thus functional integrity. These patterned cells allowed us to construct quantitative maps of the spatial organization of intracellular membranous compartments and to detect subtle changes in endomembrane organization induced by cytoskeleton disruption."

http://www.nature.com/nmeth/journal/v7/n7/full/nmeth.1462.html

"Cells were cultured on micropatterns of extracellular matrix proteins that standardize cell shape and allow alignment of CD63-marked structures. Combining the signals of CD63-marked components from several tens of cells, we showed that the 3D organization of MVB is reproducible in these normalized conditions (...) This test allowed us to compare complex data from fluorescent microscopy without reducing the provided information into simple summary statistics. This allowed quantitative comparison of cellular morphology by directly measuring the three-dimensional organization of intracellular structures visualized by fluorescent microscopy."

http://www.pnas.org/content/109/22/8382.long

"Cell culture on ECM-coated micropatterns (...) does offer the potential to manipulate the spatial distribution of cell adhesions and thereby the potential to provide mechanistic insights to cell behavior in vivo"

http://www.cell.com/cell-reports/fulltext/S2211-1247%2813%2900542-1

"The results of our studies demonstrate that single hPSC cytoskeleton and cell adhesion can be precisely controlled by ECM micropatterns. These results provide an opportunity for further investigation of hPSC division, cell-cell interactions, and to control stem cell fate".

http://www.ingentaconnect.com/content/asp/jbte/2013/00000003/00000004/art00010

"We developed a robust method to induce targeted and selective formation of hemisynapses with high statistics, by culturing neurons on micropatterned substrates coated with Nrx1b or SynCAM1"

http://www.nature.com/ncomms/2013/130812/ncomms3252/full/ncomms3252.html

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