Search

Maternal Embryonic Leucine Zipper Kinase (MELK) has been studied intensively in recent years due to its overexpression in multiple cancers. However, the cell biology of MELK remains less characterized despite its well-documented association with mitosis. Here we report a distinctive pattern of human MELK that translocates from the cytoplasm to cell cortex within 3 min of anaphase onset. The cortex association lasts about 30 min till telophase. The spatiotemporal specific localization of MELK depends on the interaction between its Threonine-Proline (TP) rich domain and kinase associated 1 (KA1) domain, which is regulated by CDK1 kinase and PP4 protein phosphatase. KA1 domains are known to regulate kinase activities through various intramolecular interactions. Our results revealed a new role for KA1 domain to control subcellular localization of a protein kinase.

The coordination between actin and microtubule network is crucial, yet this remains a challenging problem to dissect and our understanding of the underlying mechanisms remains limited. In this study, we used travelling waves in the cell cortex to characterize the collective dynamics of cytoskeletal networks. Our findings show that Cdc42 and F-BAR-dependent actin waves in mast cells are mainly driven by formin-mediated actin polymerization, with the microtubule-binding formin FH2 domain-containing protein 1 (FHDC1) as an early regulator. Knocking down FHDC1 inhibits actin wave formation, and this inhibition require FHDC1’s interaction with both microtubule and actin. The phase of microtubule depolymerization coincides with the nucleation of actin waves and microtubule stabilization inhibit actin waves, leading us to propose that microtubule shrinking and the concurrent release of FHDC1 locally regulate actin nucleation. Lastly, we show that FHDC1 is crucial for multiple cellular processes such as cell division and migration. Our data provided molecular insights into the nucleation mechanisms of actin waves and uncover an antagonistic interplay between microtubule and actin polymerization in their collective dynamics.

Introduction: Nuclei have characteristic shapes dependent on cell type, which are critical for proper cell function, and nuclei lose their distinct shapes in multiple diseases including cancer, laminopathies, and progeria. Nuclear shapes result from deformations of the sub-nuclear components—nuclear lamina and chromatin. How these structures respond to cytoskeletal forces to form the nuclear shape remains unresolved. Although the mechanisms regulating nuclear shape in human tissues are not fully understood, it is known that different nuclear shapes arise from cumulative nuclear deformations post-mitosis, ranging from the rounded morphologies that develop immediately after mitosis to the various nuclear shapes that roughly correspond to cell shape (e.g., elongated nuclei in elongated cells, flat nuclei in flat cells).Methods: We formulated a mathematical model to predict nuclear shapes of cells in various contexts under the geometric constraints of fixed cell volume, nuclear volume and lamina surface area. Nuclear shapes were predicted and compared to experiments for cells in various geometries, including isolated on a flat surface, on patterned rectangles and lines, within a monolayer, isolated in a well, or when the nucleus is impinging against a slender obstacle.Results and Discussion: The close agreement between predicted and experimental shapes demonstrates a simple geometric principle of nuclear shaping: the excess surface area of the nuclear lamina (relative to that of a sphere of the same volume) permits a wide range of highly deformed nuclear shapes under the constraints of constant surface area and constant volume. When the lamina is smooth (tensed), the nuclear shape can be predicted entirely from these geometric constraints alone for a given cell shape. This principle explains why flattened nuclear shapes in fully spread cells are insensitive to the magnitude of the cytoskeletal forces. Also, the surface tension in the nuclear lamina and nuclear pressure can be estimated from the predicted cell and nuclear shapes when the cell cortical tension is known, and the predictions are consistent with measured forces. These results show that excess surface area of the nuclear lamina is the key determinant of nuclear shapes. When the lamina is smooth (tensed), the nuclear shape can be determined purely by the geometric constraints of constant (but excess) nuclear surface area, nuclear volume, and cell volume, for a given cell adhesion footprint, independent of the magnitude of the cytoskeletal forces involved.

The formation of specific cellular protrusions, plasma membrane blebs, underlies the amoeboid mode of cell motility, which is characteristic for free-living amoebae and leukocytes, and can also be adopted by stem and tumor cells to bypass unfavorable migration conditions and thus facilitate their long-distance migration. Not all cells are equally prone to bleb formation. We have previously shown that membrane blebbing can be experimentally induced in a subset of HT1080 fibrosarcoma cells, whereas other cells in the same culture under the same conditions retain non-blebbing mesenchymal morphology. Here we show that this heterogeneity is associated with the distribution of vimentin intermediate filaments (VIFs). Using different approaches to alter the VIF organization, we show that blebbing activity is biased toward cell edges lacking abundant VIFs, whereas the VIF-rich regions of the cell periphery exhibit low blebbing activity. This pattern is observed both in interphase fibroblasts, with and without experimentally induced blebbing, and during mitosis-associated blebbing. Moreover, the downregulation of vimentin expression or displacement of VIFs away from the cell periphery promotes blebbing even in cells resistant to bleb-inducing treatments. Thus, we reveal a new important function of VIFs in cell physiology that involves the regulation of non-apoptotic blebbing essential for amoeboid cell migration and mitosis.

The cell cortex is an active viscoelastic self-deforming sheet at the periphery of animal cells. It constricts animal cells during cell division. For some egg cells, the actomyosin cortex was shown to exhibit counter-rotating chiral flows along the axis of division. Such chiral surface flows were shown to contribute to spatial rearrangements and left-right symmetry breaking in developing organisms. In spite of this prospective biological importance, the effect of chiral forces on the flows and emergent shape dynamics of a deformable surface are completely unknown. To shed a first light on that matter, we present here a numerical study of an axisymmetric viscoelastic surface embedded in a viscous fluid. We impose a generic counter-rotating force field on this surface and study the resulting chiral flow field and shape dynamics for various surface mechanical parameters. Notably, we find that the building of a neck, as is observed during cell division, occurs if the surface contains a strong shear elastic component. Furthermore, we find that a large areal relaxation time results in flows towards the equator of the surface. These flows assist the transport of a surface concentration during the formation of a contractile ring. Accordingly, we show that chiral forces by themselves can drive pattern formation and stabilise contractile rings at the equator. These results provide first mechanistic evidence that chiral flows can play a significant role to orchestrate cell division.

The cell cortex is a highly dynamic network of cytoskeletal filaments in which motor proteins induce active cortical stresses which in turn drive dynamic cellular processes such as cell motility, furrow formation or cytokinesis during cell division. Here, we develop a three-dimensional computational model of a cell cortex in the viscous limit including active cortical flows. Combining active gel and thin shell theory, we base our computational tool directly on the force balance equations for the velocity field on a discretized and arbitrarily deforming cortex. Since our method is based on the general force balance equations, it can easily be extended to more complex biological dependencies in terms of the constitutive laws or a dynamic coupling to a suspending fluid. We validate our algorithm by investigating the formation of a cleavage furrow on a biological cell immersed in a passive outer fluid, where we successfully compare our results to axi-symmetric simulations. We then apply our fully three-dimensional algorithm to fold formation and to study furrow formation under the influence of non-axisymmetric disturbances such as external shear. We report a reorientation mechanism by which the cell autonomously realigns its axis perpendicular to the furrow plane thus contributing to the robustness of cell division under realistic environmental conditions.

Shear flow-induced migration is an important physiological phenomenon experienced by multiple cell types, including leukocytes and cancer cells. However, molecular mechanisms by which cells sense and directionally migrate in response to mechanical perturbation are not well understood. Dictyostelium discoideum social amoeba, a well-established model for studying amoeboid-type migration, also exhibits directional motility when exposed to shear flow, and this behavior is preceded by rapid and transient activation of the same signal transduction network that is activated by chemoattractants. The initial response, which can also be observed following brief 2 s stimulation with shear flow, requires an intact actin cytoskeleton; however, what aspect of the cytoskeletal network is responsible for sensing and/or transmitting the signal is unclear. We investigated the role of actin crosslinkers filamin and α-actinin by analyzing initial shear flow-stimulated responses in cells with or without these proteins. Both filamin and α-actinin showed rapid and transient relocalization from the cytosol to the cortex following shear flow stimulation. Using spatiotemporal analysis of Ras GTPase activation as a readout of signal transduction network activity, we demonstrated that lack of α-actinin did not reduce, and, in fact, slightly improved the response to acute mechanical stimulation compared to cells expressing α-actinin. In contrast, shear flow-induced Ras activation was significantly more robust in filamin-null cells rescued with filamin compared to cells expressing empty vector. Reduced responsiveness appeared to be specific to mechanical stimuli and was not due to a change in the basal activity since response to global stimulation with a chemoattractant and random migration was comparable between cells with or without filamin. Finally, while filamin-null cells rescued with filamin efficiently migrated upstream when presented with continuous flow, cells lacking filamin were defective in directional migration. Overall, our study suggests that filamin, but not α-actinin, is involved in sensing and/or transmitting mechanical stimuli that drive directed migration; however, other components of the actin cytoskeleton likely also contribute to the initial response since filamin-null cells were still able to activate the signal transduction network. These findings could have implications for our fundamental understanding of shear flow-induced migration of leukocytes, cancer cells and other amoeboid-type cells.

Structures and machines require smoothening of raw materials. Self-organized smoothening guides cell and tissue morphogenesis and is relevant to advanced manufacturing. Across the syncytial Drosophila embryo surface, smooth interfaces form between expanding Arp2/3-based actin caps and surrounding actomyosin networks, demarcating the circumferences of nascent dome-like compartments used for pseudocleavage. We found that forming a smooth and circular boundary of the surrounding actomyosin domain requires Arp2/3 in vivo. To dissect the physical basis of this requirement, we reconstituted the interacting networks using node-based models. In simulations of actomyosin networks with local clearances in place of Arp2/3 domains, rough boundaries persisted when myosin contractility was low. With addition of expanding Arp2/3 network domains, myosin domain boundaries failed to smoothen, but accumulated myosin nodes and tension. After incorporating actomyosin mechanosensitivity, Arp2/3 network growth locally induced a surrounding contractile actomyosin ring that smoothened the interface between the cytoskeletal domains, an effect also evident in vivo. In this way, a smooth structure can emerge from the lateral interaction of irregular active materials.

Contractile actomyosin bundles, stress fibers, govern key cellular processes including migration, adhesion, and mechanosensing. Stress fibers are thus critical for developmental morphogenesis. The most prominent actomyosin bundles, ventral stress fibers, are generated through coalescence of pre-existing stress fiber precursors. However, whether stress fibers can assemble through other mechanisms has remained elusive. We report that stress fibers can also form without requirement of pre-existing actomyosin bundles. These structures, which we named cortical stress fibers, are embedded in the cell cortex and assemble preferentially underneath the nucleus. In this process, non-muscle myosin II pulses orchestrate the reorganization of cortical actin meshwork into regular bundles, which promote reinforcement of nascent focal adhesions, and subsequent stabilization of the cortical stress fibers. These results identify a new mechanism by which stress fibers can be generated de novo from the actin cortex and establish role for stochastic myosin pulses in the assembly of functional actomyosin bundles.

Cellular membranes belong to the most vital yet least understood biomaterials of live matter. For instance, its biomechanical requirements substantially vary across species and subcellular sites, raising the question how membranes manage to adjust to such dramatic changes. Central to its adaptability at the cell surface is the interplay between the plasma membrane and the adjacent cell cortex, forming an adaptive composite material that dynamically adjusts its mechanical properties. Using a hypothetical composite material, we identify core challenges, and discuss how cellular membranes solved these tasks. We further muse how pathological changes in material properties affect membrane mechanics and cell function, before closing with open questions and future challenges arising when studying cellular membranes.

Abstract Morphogenesis in animal tissues is largely driven by actomyosin networks, through tensions generated by an active contractile process. Although the network components and their properties are known, and networks can be reconstituted in vitro, the requirements for contractility are still poorly understood. Here, we describe a theory that predicts whether an isotropic network will contract, expand, or conserve its dimensions. This analytical theory correctly predicts the behavior of simulated networks, consisting of filaments with varying combinations of connectors, and reveals conditions under which networks of rigid filaments are either contractile or expansile. Our results suggest that pulsatility is an intrinsic behavior of contractile networks if the filaments are not stable but turn over. The theory offers a unifying framework to think about mechanisms of contractions or expansion. It provides the foundation for studying a broad range of processes involving cytoskeletal networks and a basis for designing synthetic networks.

Cellular secretion depends on exocytosis of secretory vesicles and discharge of vesicle contents. Actin and myosin are essential for pre-fusion and post-fusion stages of exocytosis. Secretory vesicles depend on actin for transport to and attachment at the cell cortex during the pre-fusion phase. Actin coats on fused vesicles contribute to stabilization of large vesicles, active vesicle contraction and/or retrieval of excess membrane during the post-fusion phase. Myosin molecular motors complement the role of actin. Myosin V is required for vesicle trafficking and attachment to cortical actin. Myosin I and II members engage in local remodeling of cortical actin to allow vesicles to get access to the plasma membrane for membrane fusion. Myosins stabilize open fusion pores and contribute to anchoring and contraction of actin coats to facilitate vesicle content release. Actin and myosin function in secretion is regulated by a plethora of interacting regulatory lipids and proteins. Some of these processes have been first described in non-neuronal cells and reflect adaptations to exocytosis of large secretory vesicles and/or secretion of bulky vesicle cargoes. Here we collate the current knowledge and highlight the role of actomyosin during distinct phases of exocytosis in an attempt to identify unifying molecular mechanisms in non-neuronal secretory cells.

Microtubules execute diverse mitotic events that are spatially and temporally separated; the underlying regulation is poorly understood. By combining drug treatments, large-scale immunoprecipitation and mass spectrometry, we report the first comprehensive map of mitotic phase-specific protein interactions of the microtubule-end binding protein, EB1. EB1 interacts with some, but not all, of its partners throughout mitosis. We show that the interaction of EB1 with Astrin-SKAP complex, a key regulator of chromosome segregation, is enhanced during prometaphase, compared to anaphase. We find that EB1 and EB3, another EB family member, can interact directly with SKAP, in an SXIP-motif dependent manner. Using an SXIP defective mutant that cannot interact with EB, we uncover two distinct pools of SKAP at spindle microtubules and kinetochores. We demonstrate the importance of SKAP's SXIP-motif in controlling microtubule growth rates and anaphase onset, without grossly disrupting spindle function. Thus, we provide the first comprehensive map of temporal changes in EB1 interactors during mitosis and highlight the importance of EB protein interactions in ensuring normal mitosis.

The actomyosin ring generates force to ingress the cytokinetic cleavage furrow in animal cells, yet its filament organization and the mechanism of contractility is not well understood. We quantified actin filament order in human cells using fluorescence polarization microscopy and found that cleavage furrow ingression initiates by contraction of an equatorial actin network with randomly oriented filaments. The network subsequently gradually reoriented actin filaments along the cell equator. This strictly depended on myosin II activity, suggesting local network reorganization by mechanical forces. Cortical laser microsurgery revealed that during cytokinesis progression, mechanical tension increased substantially along the direction of the cell equator, while the network contracted laterally along the pole-to-pole axis without a detectable increase in tension. Our data suggest that an asymmetric increase in cortical tension promotes filament reorientation along the cytokinetic cleavage furrow, which might have implications for diverse other biological processes involving actomyosin rings.