Systems Cell Biology of Cell polarity and Cell division

Keywords: cell polarity, cell division, cell migration, cytoskeleton, micro-fabrication

Our team studies the process of cell polarization, in the context of cell migration and cell division. A cell is polarized when it has developed a main axis of organization. Polarization can occur spontaneously or be triggered by external signals, like gradients of signaling molecules, light, forces or even electrical fields. It involves a reorganization of the cell cytoskeleton and intracellular organelles and eventually results in a specific cell response: directional migration or growth, oriented division, oriented secretion, etc…

The project of our team is to understand both the molecular mechanisms and physical principles governing cell polarity. To achieve that goal, we use and develop innovative tools based on micro-fabrication techniques, in order to control the main physical and chemical parameters of the cell micro-environment. These tools are coupled with high quality quantitative microscopy, as well as regular molecular and cell biology techniques, providing a quantitative description of the cell behaviour.

The originality of our approach is to integrate several areas of expertise, physical chemistry, soft matter physics and cell biology, in a single team, to answer a fundamental biological question: how cells develop a polarized behaviour, in order to provide new basic concepts and innovative tools with potential application in the biomedical field.

Fig. 1 

Controlling the micro-environment of cultured cells with micro-fabricated tools

In vivo, cells have to face a complex environment, whereas, in vitro, even if cells are more amenable to experimentation, the culture conditions are often over-simplified if not completely artefactual. Thanks to recent progress in micro-fabrication, we develop in our lab micro-tools to control cell adhesion, substrate physical properties, cell shape, etc. These techniques are also compatible with micro-fluidics, which allows us to control the chemical environment of cells (drugs, nutriments, etc).
One can expect that working in controlled micron scale environments will be a revolution comparable to light microscopy and live cell imaging over the last decade.

We are currently developing a method to obtain ‘dynamic' adhesive micropatterns, with a geometry that can be modulated at will during the experiment. This will allow us to study the dynamics of cell polarization, and to induce directed cell movements, but also to modulate the geometry of cell adhesion during division. We have been working on making micropatterns compatible with high content screening, by making 96 wells micro-patterned plates. We have managed to develop a micro-patterning technique allowing the assembly of such plates. The next step is to test them on a screening plateform and to make SiRNA and micro-patterning compatible. CYTOO, a company based in Grenoble and selling micro-patterned slides and plates based on our patented technology, has been founded in 2008.

Cell migration in confined environments

Cell migration has been studied by microscopy on flat glass substrates for years and with a great success. But recent studies showed that it is certainly not the complete picture: the cell environment in a living tissue is highly complex, involving cell/cell interactions as well as cell/matrix interactions. We need actually further strategies to understand the mechanisms of cell migration in tissue.
Systems such as collagen gels are commonly used as simplified models to study cell migration in 3-D environments. But the results are difficult to interpret: many physical parameters (gel density, unevenness, elasticity…) act simultaneously and affect the motility of cells embedded in such gels. Here we propose an innovative experimental approach based on micro-fabrication to disentangle these physical parameters and address their individual roles in cell motility in complex 3-D structures.

Fig. 2 

Similar micro-channels have been used to study morphogenesis of the yeast S. Pombe, in collaboration with the team of Phong Tran and Anne Paoletti. This work provides direct evidence that the cytoskeleton controls cell polarity and cell shape and demonstrates that cell shape also controls the organization of the cytoskeleton, in a feedback loop. We present a model of the feedback loop which explains how fission yeast cells maintain a rod-shape, and how perturbation of specific parameters of the loop can lead to different cell shapes.

Fig. 3 

The role of forces exerted on cells during their division

Evaluating forces involved in cellular processes and understanding their role is one of the main challenges of the physical approaches to cell biology. Indeed, the interaction of cells with their immediate surrounding is in part mediated by biochemical signals, but also by mechanical ones (mecanotransduction). Most of studies measuring or modifying forces have been performed in vitro and almost exclusively on interphase cells. It turns out that most often cultured cells would almost detach from the substrate when they enter mitosis, which might explain why mecanotransduction was not studied in this context. Nevertheless, they keep links with the substrate, called retraction fibers. These links have been very poorly studied and their physiological relevance have not been proven directly. However, recent studies in which our group has been involved have suggested that these fibers could provide spatial information to dividing cells, and be involved in setting the mitotic spindle orientation and thus the division axis. We also believe that mecanotransduction might be involved in the control of the very last step of cell division, called abscission, whose failure might lead to aneuploidy and cancer.

Fig. 4 

Patent

Adhesive control of internal cell organisation is an efficient and low-cost method that allows screening of genes or compounds activities on cell functions encompassing polarity, motility and division as well as internal compartmentalisation and transport.

Bornens M, Thery M and Piel M : Methods and Device for Adhesive Control of Internal Cell Organization (ACICO). Mars 2005 (PCT: WO026313), mai 2007 (Europe: 1664266), février 2007 (USA: 004283-A1), mars 2007 (Japon: 504818).

Last update: January 2009

Key publications

2009

• Ammar Azioune, Marko Storch, Michel Bornens, Manuel Théry and Matthieu Piel
  Simple and rapid process for single cell micro-patterning
  Lab On Chip, Lab Chip, 2009, 9, 1640–1642 - Abstract
  We present a simple and environmentally friendly process for cell patterning on glass covered with an
  ultrathin layer of poly-L-lysine-grafted-polyethylene glycol (PLL-g-PEG) by exposure to deep UV light.
  The patterned substrates are stable for months in the lab atmosphere before incubation with proteins.
  Incubation with proteins resulted in well defined patterns, with high feature resolution. RPE-1 cells
  seeded on fibronectin/fibrinogen–Alexa 488 patterns were constrained for days on the deep UV exposed
  regions. Finally, large glass plates were patterned with high homogeneity enabling the assembly of
  micro-patterned microplates in 96-well format.
  - Full version

2008

• Courtney RT*, Makushok T*, Baigl D, Chen Y, Bornens M, Paoletti A, Piel M*, Tran PT*
  Physical mechanisms redirecting cell polarity and cell shape
  Current Biology, 18(22):1748-53 - Abstract
  The cylindrical rod shape of the fission yeast Schizosaccharomyces pombe is organized and maintained by interactions between the microtubule, cell membrane, and actin cytoskeleton [1]. Mutations affecting any components in this pathway lead to bent, branched, or round cells [2]. In this context, the cytoskeleton controls cell polarity and thus dictates cell shape. Here, we use soft-lithography techniques to construct microfluidic channels to control cell shape. We show that when wild-type rod-shaped cells are physically forced to grow in a bent fashion, they will reorganize their cytoskeleton and redirect cell polarity to make new ectopic cell tips. Moreover, when bent or round mutant cells are physically forced to conform to the wild-type rod-shape, they will reverse their mutational phenotypes by reorganizing their cytoskeleton to maintain proper wild-type-like localization of microtubules, cell-membrane proteins, and actin. Our study provides direct evidence that the cytoskeleton controls cell polarity and cell shape and demonstrates that cell shape also controls the organization of the cytoskeleton in a feedback loop. We present a model of the feedback loop to explain how fission yeast maintain a rod shape and how perturbation of specific parameters of the loop can lead to different cell shapes.
  - Full version
• Faure-André G*, Vargas P*, Yuseff M-Y, Heuzé M, Diaz J, Lankar D, Steri V, Manry J, Hugues S, Vascotto F, Boulanger J, Raposo G, Bono M-R, Rosemblatt M, Piel M*, and Lennon-Duménil A-M*
  Regulation of Dendritic Cell Migration by CD74, the MHC class II-associated Invariant Chain
  Science, 322(5908):1705-10 - Abstract
  Dendritic cells (DCs) sample peripheral tissues of the body in search of antigens to present to T cells. This requires two processes, antigen processing and cell motility, originally thought to occur independently. We found that the major histocompatibility complex II-associated invariant chain (Ii or CD74), a known regulator of antigen processing, negatively regulates DC motility in vivo. By using microfabricated channels to mimic the confined environment of peripheral tissues, we found that wild-type DCs alternate between high and low motility, whereas Ii-deficient cells moved in a faster and more uniform manner. The regulation of cell motility by Ii depended on the actin-based motor protein myosin II. Coupling antigen processing and cell motility may enable DCs to more efficiently detect and process antigens within a defined space.
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2007

• Fink J, Thery M, Azioune A, Dupont R, Chatelain, F, Bornens M, Piel M
  Comparative Study and Improvement of Current Cell Micro-patterning Techniques
  Lab On a Chip, 7, 672 - 680, DOI: 10.1039/b618545b - Abstract
  The original micropatterning technique on gold, although very efficient, is not accessible to most biology labs and is not compatible with their techniques for image acquisition. Other solutions have been developed on silanized glass coverslips. These methods are still hardly accessible to biology labs and do not provide sufficient reproducibility to become incorporated in routine biological protocols. Here, we analyzed cell behavior on micro-patterns produced by various alternative techniques. Distinct cell types displayed different behavior on micropatterns, while some were easily constrained by the patterns others escaped or ripped off the patterned adhesion molecules. We report methods to overcome some of these limitations on glass coverslips and on plastic dishes which are compatible with our experimental biological applications. Finally, we present a new method based on UV crosslinking of adhesion proteins with benzophenone to easily and rapidly produce highly reproducible micropatterns without the use of a microfabricated elastomeric stamp.
  - Full version

2006

• Thery M, Racine V, Piel M, Pepin A, Dimitrov A, Chen Y, Sibarita JB, Bornens M.
  Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity
  Proc Natl Acad Sci U S A, Dec 26;103(52):19771-6 - Abstract
  Control of the establishment of cell polarity is an essential function in tissue morphogenesis and renewal that depends on spatial cues provided by the extracellular environment. The molecular role of cell-cell or cell-extracellular matrix (ECM) contacts on the establishment of cell polarity has been well characterized. It has been hypothesized that the geometry of the cell adhesive microenvironment was directing cell surface polarization and internal organization. To define how the extracellular environment affects cell polarity, we analyzed the organization of individual cells plated on defined micropatterned substrates imposing cells to spread on various combinations of adhesive and nonadhesive areas. The reproducible normalization effect on overall cell compartmentalization enabled quantification of the spatial organization of the actin network and associated proteins, the spatial distribution of microtubules, and the positioning of nucleus, centrosome, and Golgi apparatus. By using specific micropatterns and statistical analysis of cell compartment positions, we demonstrated that ECM geometry determines the orientation of cell polarity axes. The nucleus-centrosome orientations were reproducibly directed toward cell adhesive edges. The anisotropy of the cell cortex in response to the adhesive conditions did not affect the centrosome positioning at the cell centroid. Based on the quantification of microtubule plus end distribution we propose a working model that accounts for that observation. We conclude that, in addition to molecular composition and mechanical properties, ECM geometry plays a key role in developmental processes.
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2005

• Jiang X, Bruzewicz DA, Wong AP, Piel M, Whitesides GM.
  Directing cell migration with asymmetric micropatterns
  Proc Natl Acad Sci U S A., Jan 25;102(4):975-8 - Abstract
  This report shows that the direction of polarization of attached mammalian cells determines the direction in which they move. Surfaces micropatterned with appropriately functionalized self-assembled monolayers constrain individual cells to asymmetric geometries (for example, a teardrop); these geometries polarize the morphology of the cell. After electrochemical desorption of the self-assembled monolayers removes these constraints and allows the cells to move across the surface, they move toward their blunt ends.
  - Full version
• Thery M, Racine V, Pepin A, Piel M, Chen Y, Sibarita JB, Bornens M.
  The extracellular matrix guides the orientation of the cell division axis
  Nat Cell Biol., Oct;7(10):947-53 - Abstract
  The cell division axis determines the future positions of daughter cells and is therefore critical for cell fate. The positioning of the division axis has been mostly studied in systems such as embryos or yeasts, in which cell shape is well defined. In these cases, cell shape anisotropy and cell polarity affect spindle orientation. It remains unclear whether cell geometry or cortical cues are determinants for spindle orientation in mammalian cultured cells. The cell environment is composed of an extracellular matrix (ECM), which is connected to the intracellular actin cytoskeleton via transmembrane proteins. We used micro-contact printing to control the spatial distribution of the ECM on the substrate and demonstrated that it has a role in determining the orientation of the division axis of HeLa cells. On the basis of our analysis of the average distributions of actin-binding proteins in interphase and mitosis, we propose that the ECM controls the location of actin dynamics at the membrane, and thus the segregation of cortical components in interphase. This segregation is further maintained on the cortex of mitotic cells and used for spindle orientation.
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2002

• Chevrier V*, Piel M*, Collomb N, Saoudi Y, Frank R, Paintrand M, Narumiya S, Bornens M, Job D.(*co-auteurs)
  The Rho-associated protein kinase p160ROCK is required for centrosome positioning
  J Cell Biol., May 27;157(5):807-17 - Abstract
  The p160-Rho-associated coiled-coil-containing protein kinase (ROCK) is identified as a new centrosomal component. Using immunofluorescence with a variety of p160ROCK antibodies, immuno EM, and depletion with RNA interference, p160ROCK is principally bound to the mother centriole (MC) and an intercentriolar linker. Inhibition of p160ROCK provoked centrosome splitting in G1 with the MC, which is normally positioned at the cell center and shows little motion during G1, displaying wide excursions around the cell periphery, similar to its migration toward the midbody during cytokinesis. p160ROCK inhibition late after anaphase in mitosis triggered MC migration to the midbody followed by completion of cell division. Thus, p160ROCK is required for centrosome positioning and centrosome-dependent exit from mitosis.
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2001

• Piel M, Nordberg J, Euteneuer U, Bornens M.
  Centrosome-dependent exit of cytokinesis in animal cells
  Science, Feb 23;291(5508):1550-3 - Abstract
  As an organelle coupling nuclear and cytoplasmic divisions, the centrosome is essential to mitotic fidelity, and its inheritance could be critical to understanding cell transformation. Investigating the behavior of the centrosome in living mitotic cells, we documented a transient and remarkable postanaphase repositioning of this organelle, which apparently controls the release of central microtubules from the midbody and the completion of cell division. We also observed that the absence of the centrosome leads to defects in cytokinesis. Together with recent results in yeasts, our data point to a conserved centrosome-dependent pathway that integrates spatial controls into the decision of completing cell division, which requires the repositioning of the centrosome organelle.
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2000

• Piel M, Meyer P, Khodjakov A, Rieder CL, Bornens M.
  The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrate cells
  Journal of Cell Biology, Apr 17;149(2):317-30 - Abstract
  We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts). or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G1/S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated gamma-tubulin, and nucleate similar number of Mts in Mt repolymerization experiments. during G1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.
  - Full version