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Metastasis is complex, involving multiple genetic, epigenetic, biochemical, and physical changes in the cancer cell and its microenvironment. Cells with metastatic potential are often characterized by altered cellular contractility and deformability, lending them the flexibility to disseminate and navigate through different microenvironments. We demonstrate that mechanoresponsiveness is a hallmark of pancreatic cancer cells. Key mechanoresponsive proteins, those that accumulate in response to mechanical stress, specifically nonmuscle myosin IIA (MYH9) and IIC (MYH14), α-actinin 4, and filamin B, were highly expressed in pancreatic cancer as compared with healthy ductal epithelia. Their less responsive sister paralogs—myosin IIB (MYH10), α-actinin 1, and filamin A—had lower expression differential or disappeared with cancer progression. We demonstrate that proteins whose cellular contributions are often overlooked because of their low abundance can have profound impact on cell architecture, behavior, and mechanics. Here, the low abundant protein MYH14 promoted metastatic behavior and could be exploited with 4-hydroxyacetophenone (4-HAP), which increased MYH14 assembly, stiffening cells. As a result, 4-HAP decreased dissemination, induced cortical actin belts in spheroids, and slowed retrograde actin flow. 4-HAP also reduced liver metastases in human pancreatic cancer-bearing nude mice. Thus, increasing MYH14 assembly overwhelms the ability of cells to polarize and invade, suggesting targeting the mechanoresponsive proteins of the actin cytoskeleton as a new strategy to improve the survival of patients with pancreatic cancer. Significance: This study demonstrates that mechanoresponsive proteins become upregulated with pancreatic cancer progression and that this system of proteins can be pharmacologically targeted to inhibit the metastatic potential of pancreatic cancer cells.

Chronic obstructive pulmonary disease (COPD) is characterized by the destruction of alveolar tissue (in emphysema) and airway remodeling (leading to chronic bronchitis), which cause difficulties in breathing. It is a growing public health concern with few therapeutic options that can reverse disease progression or mortality. This is in part because current treatments mainly focus on ameliorating symptoms induced by inflammatory pathways as opposed to curing disease. Hence, emerging research focused on upstream pathways are likely to be beneficial in the development of efficient therapeutics to address the root causes of disease. Some of these pathways include mitochondrial function, cytoskeletal structure and maintenance, and airway hydration, which are all affected by toxins that contribute to COPD. Because of the complexity of COPD and unknown targets for disease onset, simpler model organisms have proved to be useful tools in identifying disease-relevant pathways and targets. This review summarizes COPD pathology, current treatments, and therapeutic discovery research, with a focus on the aforementioned pathways that can advance the therapeutic landscape of COPD.

Cancer progression is dependent on heightened mechanical adaptation, both for the cells’ ability to change shape and to interact with varying mechanical environments. This type of adaptation is dependent on mechanoresponsive proteins that sense and respond to mechanical stress, as well as their regulators. Mechanoresponsive proteins are part of the mechanobiome, which is the larger network that constitutes the cell’s mechanical systems that are also highly integrated with many other cellular systems, such as gene expression, metabolism, and signaling. Despite the altered expression patterns of key mechanobiome proteins across many different cancer types, pharmaceutical targeting of these proteins has been overlooked. Here, we review the biochemistry of key mechanoresponsive proteins, specifically nonmuscle myosin II, α-actinins, and filamins, as well as the partnering proteins 14-3-3 and CLP36. We also examined a wide range of data sets to assess how gene and protein expression levels of these proteins are altered across many different cancer types. Finally, we determined the potential of targeting these proteins to mitigate invasion or metastasis and suggest that the mechanobiome is a goldmine of opportunity for anticancer drug discovery and development.

Schiffhauer et al. use mathematical modeling of Dictyostelium myosin II and experimental approaches in mammalian cells to demonstrate that multiple inputs modulate the ability of myosin II to assemble into filaments. These inputs integrate to govern the myosin II mechanoresponsiveness., Dynamical cell shape changes require a highly sensitive cellular system that can respond to chemical and mechanical inputs. Myosin IIs are key players in the cell’s ability to react to mechanical inputs, demonstrating an ability to accumulate in response to applied stress. Here, we show that inputs that influence the ability of myosin II to assemble into filaments impact the ability of myosin to respond to stress in a predictable manner. Using mathematical modeling for Dictyostelium myosin II, we predict that myosin II mechanoresponsiveness will be biphasic with an optimum established by the percentage of myosin II assembled into bipolar filaments. In HeLa and NIH 3T3 cells, heavy chain phosphorylation of NMIIB by PKCζ, as well as expression of NMIIA, can control the ability of NMIIB to mechanorespond by influencing its assembly state. These data demonstrate that multiple inputs to the myosin II assembly state integrate at the level of myosin II to govern the cellular response to mechanical inputs.

Spectrin is a membrane skeletal protein best known for its structural role in maintaining cell shape and protecting cells from mechanical damage. Here, we report that α/βH-spectrin (βH is also called karst) dynamically accumulates and dissolves at the fusogenic synapse between fusing Drosophila muscle cells, where an attacking fusion partner invades its receiving partner with actin-propelled protrusions to promote cell fusion. Using genetics, cell biology, biophysics and mathematical modelling, we demonstrate that spectrin exhibits a mechanosensitive accumulation in response to shear deformation, which is highly elevated at the fusogenic synapse. The transiently accumulated spectrin network functions as a cellular fence to restrict the diffusion of cell-adhesion molecules and a cellular sieve to constrict the invasive protrusions, thereby increasing the mechanical tension of the fusogenic synapse to promote cell membrane fusion. Our study reveals a function of spectrin as a mechanoresponsive protein and has general implications for understanding spectrin function in dynamic cellular processes.

The 14-3-3 family comprises a group of small proteins that are essential, ubiquitous, and highly conserved across eukaryotes. Overexpression of the 14-3-3 proteins σ, ϵ, ζ, and η correlates with high metastatic potential in multiple cancer types. In Dictyostelium, 14-3-3 promotes myosin II turnover in the cell cortex and modulates cortical tension, cell shape, and cytokinesis. In light of the important roles of 14-3-3 proteins across a broad range of eukaryotic species, we sought to determine how 14-3-3 proteins interact with myosin II. Here, conducting in vitro and in vivo studies of both Dictyostelium (one 14-3-3 and one myosin II) and human proteins (seven 14-3-3s and three nonmuscle myosin IIs), we investigated the mechanism by which 14-3-3 proteins regulate myosin II assembly. Using in vitro assembly assays with purified myosin II tail fragments and 14-3-3, we demonstrate that this interaction is direct and phosphorylation-independent. All seven human 14-3-3 proteins also altered assembly of at least one paralog of myosin II. Our findings indicate a mechanism of myosin II assembly regulation that is mechanistically conserved across a billion years of evolution from amebas to humans. We predict that altered 14-3-3 expression in humans inhibits the tumor suppressor myosin II, contributing to the changes in cell mechanics observed in many metastatic cancers.

Significance There is a pressing need for new approaches to combat metastatic disease. We demonstrate, here, a strategy that targets and activates the molecular machines that control cell shape in cell division, wound healing, immune surveillance, embryonic development, and cancer metastasis. Cells control their shape by remodeling their cytoskeletal actin filaments, microtubules, and intermediate filaments, while cytoskeletal motor proteins, such as the myosins generate forces that can produce local contractions. By targeting and activating NM2C directly, we interfere with cytoskeletal plasticity. We demonstrate the effectiveness of this activation strategy in vitro and in vivo and provide a molecular mechanism for our measured increase in cell stiffness. Our strategy can be integrated readily with existing approaches to combat aggressive cancers.

The cation channel transient receptor potential vanilloid 4 (TRPV4) is one of the few identified ion channels that can directly cause inherited neurodegeneration syndromes, but the molecular mechanisms are unknown. Here, we show that in vivo expression of a neuropathy-causing TRPV4 mutant (TRPV4

The ability of glioblastoma to disperse through the brain contributes to its lethality, and blocking this behavior has been an appealing therapeutic approach. Although a number of proinvasive signaling pathways are active in glioblastoma, many are redundant, so targeting one can be overcome by activating another. However, these pathways converge on nonredundant components of the cytoskeleton, and we have shown that inhibiting one of these-the myosin II family of cytoskeletal motors-blocks glioblastoma invasion even with simultaneous activation of multiple upstream promigratory pathways. Myosin IIA and IIB are the most prevalent isoforms of myosin II in glioblastoma, and we now show that codeleting these myosins markedly impairs tumorigenesis and significantly prolongs survival in a rodent model of this disease. However, while targeting just myosin IIA also impairs tumor invasion, it surprisingly increases tumor proliferation in a manner that depends on environmental mechanics. On soft surfaces myosin IIA deletion enhances ERK1/2 activity, while on stiff surfaces it enhances the activity of NFκB, not only in glioblastoma but in triple-negative breast carcinoma and normal keratinocytes as well. We conclude myosin IIA suppresses tumorigenesis in at least two ways that are modulated by the mechanics of the tumor and its stroma. Our results also suggest that inhibiting tumor invasion can enhance tumor proliferation and that effective therapy requires targeting cellular components that drive both proliferation and invasion simultaneously.

For specialized cell function, as well as active cell behaviors such as division, migration, and tissue development, cells must undergo dynamic changes in shape. To complete these processes, cells integrate chemical and mechanical signals to direct force production. This mechanochemical integration allows for the rapid production and adaptation of leading-edge machinery in migrating cells, the invasion of one cell into another during cell-cell fusion, and the force-feedback loops that ensure robust cytokinesis. A quantitative understanding of cell mechanics coupled with protein dynamics has allowed us to account for furrow ingression during cytokinesis, a model cell-shape-change process. At the core of cell-shape changes is the ability of the cell's machinery to sense mechanical forces and tune the force-generating machinery as needed. Force-sensitive cytoskeletal proteins, including myosin II motors and actin cross-linkers such as α -actinin and filamin, accumulate in response to internally generated and externally imposed mechanical stresses, endowing the cell with the ability to discern and respond to mechanical cues. The physical theory behind how these proteins display mechanosensitive accumulation has allowed us to predict paralog-specific behaviors of different cross-linking proteins and identify a zone of optimal actin-binding affinity that allows for mechanical stress-induced protein accumulation. These molecular mechanisms coupled with the mechanical feedback systems ensure robust shape changes, but if they go awry, they are poised to promote disease states such as cancer cell metastasis and loss of tissue integrity.

The Hippo pathway effector Yes-associated protein (YAP) regulates liver size by promoting cell proliferation and inhibiting apoptosis. However, recent in vivo studies suggest that YAP has important cellular functions other than controlling proliferation and apoptosis. Transgenic YAP expression in mouse hepatocytes results in severe jaundice. A possible explanation for the jaundice could be defects in adherens junctions that prevent bile from leaking into the blood stream. Indeed, immunostaining of E-cadherin and electron microscopic examination of bile canaliculi of Yap transgenic livers revealed abnormal adherens junction structures. Using primary hepatocytes from Yap transgenic livers and Yap knockout livers, we found that YAP antagonizes E-cadherin-mediated cell-cell junction assembly by regulating the cellular actin architecture, including its mechanical properties (elasticity and cortical tension). Mechanistically, we found that YAP promoted contractile actin structure formation by upregulating nonmuscle myosin light chain expression and cellular ATP generation. Thus, by modulating actomyosin organization, YAP may influence many actomyosin-dependent cellular characteristics, including adhesion, membrane protrusion, spreading, morphology, and cortical tension and elasticity, which in turn determine cell differentiation and tissue morphogenesis.

STUDY HYPOTHESIS Cellular aging of the egg following ovulation, also known as post-ovulatory aging, is associated with aberrant cortical mechanics and actomyosin cytoskeleton functions. STUDY FINDING Post-ovulatory aging is associated with dysfunction of non-muscle myosin-II, and pharmacologically induced myosin-II dysfunction produces some of the same deficiencies observed in aged eggs. WHAT IS KNOWN ALREADY Reproductive success is reduced with delayed fertilization and when copulation or insemination occurs at increased times after ovulation. Post-ovulatory aged eggs have several abnormalities in the plasma membrane and cortex, including reduced egg membrane receptivity to sperm, aberrant sperm-induced cortical remodeling and formation of fertilization cones at the site of sperm entry, and reduced ability to establish a membrane block to prevent polyspermic fertilization. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Ovulated mouse eggs were collected at 21-22 h post-human chorionic gonadotrophin (hCG) (aged eggs) or at 13-14 h post-hCG (young eggs), or young eggs were treated with the myosin light chain kinase (MLCK) inhibitor ML-7, to test the hypothesis that disruption of myosin-II function could mimic some of the effects of post-ovulatory aging. Eggs were subjected to various analyses. Cytoskeletal proteins in eggs and parthenogenesis were assessed using fluorescence microscopy, with further analysis of cytoskeletal proteins in immunoblotting experiments. Cortical tension was measured through micropipette aspiration assays. Egg membrane receptivity to sperm was assessed in in vitro fertilization (IVF) assays. Membrane topography was examined by low-vacuum scanning electron microscopy (SEM). MAIN RESULTS AND THE ROLE OF CHANCE Aged eggs have decreased levels and abnormal localizations of phosphorylated myosin-II regulatory light chain (pMRLC; P = 0.0062). Cortical tension, which is mediated in part by myosin-II, is reduced in aged mouse eggs when compared with young eggs, by ∼40% in the cortical region where the metaphase II spindle is sequestered and by ∼50% in the domain to which sperm bind and fuse (P < 0.0001). Aging-associated parthenogenesis is partly rescued by treating eggs with a zinc ionophore (P = 0.003), as is parthenogenesis induced by inhibition of mitogen-activated kinase (MAPK) 3/1 [also known as extracellular signal-regulated kinase (ERK)1/2] or MLCK. Inhibition of MLCK with ML-7 also results in effects that mimic those of post-ovulatory aging: fertilized ML-7-treated eggs show both impaired fertilization and increased extents of polyspermy, and ML-7-treated young eggs have several membrane abnormalities that are shared by post-ovulatory aged eggs. LIMITATIONS, REASONS FOR CAUTION These studies were done with mouse oocytes, and it remains to be fully determined how these findings from mouse oocytes would compare with other species. For studies using methods not amenable to analysis of large sample sizes and data are limited to what images one can capture (e.g. SEM), data should be interpreted conservatively. WIDER IMPLICATIONS OF THE FINDINGS These data provide insights into causes of reproductive failures at later post-copulatory times. LARGE SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This project was supported by R01 HD037696 and R01 HD045671 from the NIH to J.P.E. Cortical tension studies were supported by R01 GM66817 to D.N.R. The authors declare there are no financial conflicts of interest.

Just as it is important to understand the cell biology of signaling pathways, it is valuable also to understand mechanical forces in cells. The field of mechanobiology has a rich history, including study of cellular mechanics during mitosis and meiosis in echinoderm oocytes and zygotes dating back to the 1930s. This chapter addresses the use of micropipette aspiration (MPA) to assess cellular mechanics, specifically cortical tension, in mammalian oocytes.

Human carcinomas are comprised of complex mixtures of tumor cells that are known to compete indirectly for nutrients and growth factors. Whether tumor cells could also compete directly, for example by elimination of rivals, is not known. Here we show that human cells can directly compete by a mechanism of engulfment called entosis. By entosis, cells are engulfed, or cannibalized while alive, and subsequently undergo cell death. We find that the identity of engulfing (“winner”) and engulfed (“loser”) cells is dictated by mechanical deformability controlled by RhoA and actomyosin, where tumor cells with high deformability preferentially engulf and outcompete neighboring cells with low deformability in heterogeneous populations. We further find that activated Kras and Rac signaling impart winner status to cells by downregulating contractile myosin, allowing for the internalization of neighboring cells that eventually undergo cell death. Finally, we compute the energy landscape of cell-in-cell formation, demonstrating that a mechanical differential between winner and loser cells is required for entosis to proceed. These data define a mechanism of competition in mammalian cells that occurs in human tumors.

Cytokinesis shape change occurs through the interfacing of three modules, cell mechanics, myosin II-mediated contractile stress generation and sensing, and a control system of regulatory proteins, which together ensure flexibility and robustness. This integrated system then defines the stereotypical shape changes of successful cytokinesis, which occurs under a diversity of mechanical contexts and environmental conditions.

Recent work shows that cytokinesis and other cellular morphogenesis events are tuned by an interplay among biochemical signals, cell shape, and cellular mechanics. In cytokinesis, this includes cross-talk between the cortical cytoskeleton and the mitotic spindle in coordination with cell cycle control, resulting in characteristic changes in cellular morphology and mechanics through metaphase and cytokinesis. The changes in cellular mechanics affect not just overall cell shape, but also mitotic spindle morphology and function. This review will address how these principles apply to oocytes undergoing the asymmetric cell divisions of meiosis I and II. The biochemical signals that regulate cell cycle timing during meiotic maturation and egg activation are crucial for temporal control of meiosis. Spatial control of the meiotic divisions is also important, ensuring that the chromosomes are segregated evenly and that meiotic division is clearly asymmetric, yielding two daughter cells - oocyte and polar body - with enormous volume differences. In contrast to mitotic cells, the oocyte does not undergo overt changes in cell shape with its progression through meiosis, but instead maintains a relatively round morphology with the exception of very localized changes at the time of polar body emission. Placement of the metaphase-I and -II spindles at the oocyte periphery is clearly important for normal polar body emission, although this is likely not the only control element. Here, consideration is given to how cellular mechanics could contribute to successful mammalian female meiosis, ultimately affecting egg quality and competence to form a healthy embryo.

During cytokinesis, the cell employs various molecular machineries to separate into two daughters. Many signaling pathways are required to ensure temporal and spatial coordination of the molecular and mechanical events. Cells can also coordinate division with neighboring cells to maintain tissue integrity and flexibility. In this review, we focus on recent advances in the understanding of the molecular underpinnings of cytokinesis.

Cells activate signaling pathways at the site closest to the chemoattractant source that lead to pseudopod formation and directional movement up the gradient. We demonstrate that cytoskeletal components required for cortical tension, including MyoII and IQGAP/cortexillins help regulate the level and timing of leading-edge pathways., In response to directional stimulation by a chemoattractant, cells rapidly activate a series of signaling pathways at the site closest to the chemoattractant source that leads to F-actin polymerization, pseudopod formation, and directional movement up the gradient. Ras proteins are major regulators of chemotaxis in Dictyostelium; they are activated at the leading edge, are required for chemoattractant-mediated activation of PI3K and TORC2, and are one of the most rapid responders, with activity peaking at ∼3 s after stimulation. We demonstrate that in myosin II (MyoII) null cells, Ras activation is highly extended and is not restricted to the site closest to the chemoattractant source. This causes elevated, extended, and spatially misregulated activation of PI3K and TORC2 and their effectors Akt/PKB and PKBR1, as well as elevated F-actin polymerization. We further demonstrate that disruption of specific IQGAP/cortexillin complexes, which also regulate cortical mechanics, causes extended activation of PI3K and Akt/PKB but not Ras activation. Our findings suggest that MyoII and IQGAP/cortexillin play key roles in spatially and temporally regulating leading-edge activity and, through this, the ability of cells to restrict the site of pseudopod formation.

Summary Introduction Contractile networks are fundamental to many cellular functions, particularly cytokinesis and cell motility. Contractile networks depend on myosin-II mechanochemistry to generate sliding force on the actin polymers. However, to be contractile, the networks must also be crosslinked by crosslinking proteins, and to change the shape of the cell, the network must be linked to the plasma membrane. Discerning how this integrated network operates is essential for understanding cytokinesis contractility and shape control. Here, we analyzed the cytoskeletal network that drives furrow ingression in Dictyostelium . Results We establish that the actin polymers are assembled into a meshwork and that myosin-II does not assemble into a discrete ring in the Dictyostelium cleavage furrow of adherent cells. We show that myosin-II generates regional mechanics by increasing cleavage furrow stiffness and slows furrow ingression during late cytokinesis as compared to myoII nulls. Actin crosslinkers dynacortin and fimbrin similarly slow furrow ingression and contribute to cell mechanics in a myosin-II-dependent manner. By using FRAP, we show that the actin crosslinkers have slower kinetics in the cleavage furrow cortex than in the pole, that their kinetics differ between wild-type and myoII null cells, and that the protein dynamics of each crosslinker correlate with its impact on cortical mechanics. Conclusions These observations suggest that myosin-II along with actin crosslinkers establish local cortical tension and elasticity, allowing for contractility independent of a circumferential cytoskeletal array. Furthermore, myosin-II and actin crosslinkers may influence each other as they modulate the dynamics and mechanics of cell-shape change.

Cytokinesis, the final step of cell division, is a great example of robust cell shape regulation. A wide variety of cells ranging from the unicellular Dictyostelium to human cells in tissues proceed through highly similar, stereotypical cell shape changes during cell division. Typically, cells first round up forming a cleavage furrow in the middle, which constricts resulting in the formation of two daughter cells. Tight control of cytokinesis is essential for proper segregation of genetic and cellular materials, and its failure is deleterious to cell viability. Thus, biological systems have developed elaborate mechanisms to ensure high fidelity of cytokinesis, including the existence of multiple biochemical and mechanical pathways regulated through feedback. In this review, we focus on the built-in redundancy of the cytoskeletal machinery that allows cells to divide successfully in a variety of biological and mechanical contexts. Using Dictyostelium cytokinesis as an example, we demonstrate that the crosstalk between biochemical and mechanical signaling through feedback ensures correct assembly and function of the cell division machinery.

Recent advances are revealing quantitative aspects of cytokinesis. Further, genetic analyses and cell imaging are providing insights into the molecular dynamics of cleavage furrow ingression as well as further refining our knowledge of the zones of the mitotic spindle that regulate the contractile properties of the overlying cortex. Ultimately, however, cortical mechanics are the result of signals that emanate from the mitotic spindle. A genuine quantitative understanding of cytokinesis must include a thorough analysis of the mechanical properties of the cortex and how signals modify these properties to dictate a well-controlled, error-free cytokinesis.

Dynacortin is a novel protein that was discovered in a genetic suppressor screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line devoid of the cleavage furrow actin bundling protein, cortexillin I. While dynacortin is highly enriched in the cortex, particularly in cell-surface protrusions, it is excluded from the cleavage furrow cortex during cytokinesis. Here, we describe the biochemical characterization of this new protein. Purified dynacortin is an 80-kDa dimer with a large 5.7-nm Stokes radius. Dynacortin cross-links actin filaments into parallel arrays with a mole ratio of one dimer to 1.3 actin monomers and a 3.1 microm K(d). Using total internal reflection fluorescence microscopy, GFP-dynacortin and the actin bundling protein coronin-GFP are seen to concentrate in highly dynamic cortical structures with assembly and disassembly half-lives of about 15 s. These results indicate that cells have evolved different actin-filament cross-linking proteins with complementary cellular distributions that collaborate to orchestrate complex cell shape changes.

Vertebrate eggs are arrested at metaphase of meiosis II, a state classically known as cytostatic factor arrest. Maintenance of this arrest until the time of fertilization and then fertilization-induced exit from metaphase II are crucial for reproductive success. Another key aspect of this meiotic arrest and exit is regulation of the metaphase II spindle, which must be appropriately localized adjacent to the egg cortex during metaphase II and then progress into successful asymmetric cytokinesis to produce the second polar body. This study examined the mitogen-activated protein kinases MAPK3 and MAPK1 (also known as ERK1/2) as regulators of these two related aspects of mammalian egg biology, specifically testing whether this MAPK pathway affected myosin-II function and whether myosin-II perturbation would produce some of the same effects as MAPK pathway perturbation. Inhibition of the MEK1/2-MAPK pathway with U0126 leads to reduced levels of phosphorylated myosin-regulatory light chain (pMRLC) and causes a reduction in cortical tension, effects that are mimicked by treatment with the myosin light chain kinase (MLCK) inhibitor ML-7. These data indicate that one mechanism by which the MAPK pathway acts in eggs is by affecting myosin-II function. We further show that MAPK or MLCK inhibition induces loss of normal cortical spindle localization or parthenogenetic egg activation. This parthenogenesis is dependent on cytosolic and extracellular calcium and can be rescued by hyperloading eggs with zinc, suggesting that these effects of inhibition of MLCK or the MAPK pathway are linked with dysregulation of ion homeostasis.

We have developed a system for performing interaction genetics in Dictyostelium discoideum that uses a cDNA library complementation/multicopy suppression strategy. Chemically mutagenized cells were screened for cytokinesis-deficient mutants and one mutant was subjected to library complementation. Isolates of four different genes were recovered as modifiers of this strain's cytokinesis defect. These include the cleavage furrow protein cortexillin I, a novel protein we named dynacortin, an ezrin-radixin-moesin-family protein, and coronin. The cortexillin I locus and transcript were found to be disrupted in the strain, identifying it as the affected gene. Dynacortin is localized partly to the cell cortex and becomes enriched in protrusive regions, a localization pattern that is similar to coronin and partly dependent on RacE. During cytokinesis, dynacortin is found in the cortex and is somewhat enriched at the poles. Furthermore, it appears to be reduced in the cleavage furrow. The genetic interactions and the cellular distributions of the proteins suggest a hypothesis for cytokinesis in which the contraction of the medial ring is a function of spatially restricted cortexillin I and myosin II and globally distributed dynacortin, coronin, and RacE.

SummaryMechanosensation is emerging as a general principle of myosin motors. As demonstrated in a recent study, the single-headed myosin I molecule is an exquisite mechanosensor, able to sense strain over a very small force range.

▪ Abstract The Drosophila ovary provides a favorable model system in which to study cellular morphogenesis. The development of a mature egg involves a syncytium of 16 germline cells and over 1000 somatically derived follicle cells. Intercellular transport, stable intercellular bridges, cell migrations, cell shape changes, and specific subcellular localization of many embryonic patterning determinants contribute to egg development and require a dynamic cytoskeleton. We discuss many of the recent genetic and cell biological studies that have led to insights into how the actin cytoskeleton is assembled and regulated during the morphogenesis of the Drosophila egg.

Drosophila kelch has four protein domains, two of which are found in kelch-family proteins and in numerous nonkelch proteins. In Drosophila, kelch is required to maintain ring canal organization during oogenesis. We have performed a structure–function analysis to study the function of Drosophila kelch. The amino-terminal region (NTR) regulates the timing of kelch localization to the ring canals. Without the NTR, the protein localizes precociously and destabilizes the ring canals and the germ cell membranes, leading to dominant sterility. The amino half of the protein including the BTB domain mediates dimerization. Oligomerization through the amino half of kelch might allow cross-linking of ring canal actin filaments, organizing the inner rim cytoskeleton. The kelch repeat domain is necessary and sufficient for ring canal localization and likely mediates an additional interaction, possibly with actin.

In Drosophila oogenesis, the development of a mature oocyte depends on having properly developed ring canals that allow cytoplasm transport from the nurse cells to the oocyte. Ring canal assembly is a step-wise process that transforms an arrested cleavage furrow into a stable intercellular bridge by the addition of several proteins. Here we describe a new gene we named cheerio that provides a critical function for ring canal assembly. Mutants in cheerio fail to localize ring canal inner rim proteins including filamentous actin, the ring canal-associated products from the hu-li tai shao (hts) gene, and kelch. Since hts and kelch are present but unlocalized in cheerio mutant cells, cheerio is likely to function upstream from each of them. Examination of mutants in cheerio places it in the pathway of ring canal assembly between cleavage furrow arrest and localization of hts and actin filaments. Furthermore, this mutant reveals that the inner rim cytoskeleton is required for expansion of the ring canal opening and for plasma membrane stabilization.

Changes occurring as the prophase I oocyte matures to metaphase II are critical for the acquisition of competence for normal egg activation and early embryogenesis. A prophase I oocyte cannot respond to a fertilizing sperm as a metaphase II egg does, including the ability to prevent polyspermic fertilization. Studies here demonstrate that the competence for the membrane block to polyspermy is deficient in prophase I mouse oocytes. In vitro fertilization experiments using identical insemination conditions result in monospermy in 87% of zona pellucida (ZP)-free metaphase II eggs, while 92% of ZP-free prophase I oocytes have four or more fused sperm. The membrane block is associated with a postfertilization reduction in the capacity to support sperm binding, but this reduction in sperm-binding capacity is both less robust and slower to develop in fertilized prophase I oocytes. Fertilization of oocytes is dependent on the tetraspanin CD9, but little to no release of CD9 from the oocyte membrane is detected, suggesting that release of CD9-containing vesicles is not essential for fertilization. The deficiency in membrane block establishment in prophase I oocytes correlates with abnormalities in two postfertilization cytoskeletal changes: sperm-induced cortical remodeling that results in fertilization cone formation and a postfertilization increase in effective cortical tension. These data indicate that cortical maturation is a component of cytoplasmic maturation during the oocyte-to-egg transition and that the egg cortex has to be appropriately primed and tuned to be responsive to a fertilizing sperm.

We analyzed the structure of cytoplasmic bridges called ring canals in Drosophila egg chambers. Two mutations, hu-li tai shao (hts) and kelch, disrupt normal ring canal development. We raised antibodies against the carboxyterminal tail of hts and found that they recognize a protein that localizes specifically to ring canals very early in ring canal assembly. Accumulation of filamentous actin on ring canals coincides with the appearance of the hts protein. kelch, which is localized to the ring canals hours after hts and actin, is necessary for maintaining a highly ordered ring canal rim since kelch mutant egg chambers have ring canals that are obstructed by disordered actin and hts. Anti-phosphotyrosine antibodies immunostain ring canals beginning early in the germarium before hts and actin and throughout egg chamber development. The use of antibody reagents to analyze the structure of wild-type and mutant ring canals has shown that ring canal development is a dynamic process of cytoskeletal protein assembly, possibly regulated by tyrosine phosphorylation of some ring canal components.

Cytokinesis is emerging as a control system defined by interacting biochemical and mechanical modules, which form a system of feedback loops. This integrated system accounts for the regulation and kinetics of cytokinesis furrowing and demonstrates that cytokinesis is a whole-cell process in which the global and equatorial cortices and cytoplasm are active players in the system. Though originally defined in Dictyostelium, features of the control system are recognizable in other organisms, suggesting a universal mechanism for cytokinesis regulation and contractility.

Background The ability of a cell to change shape is crucial for the proper function of many cellular processes, including cell migration. One type of cell migration, referred to as amoeboid motility, involves alternating cycles of morphological expansion and retraction. Traditionally, this process has been characterized by a number of parameters providing global information about shape changes, which are insufficient to distinguish phenotypes based on local pseudopodial activities that typify amoeboid motility. Results We developed a method that automatically detects and characterizes pseudopodial behavior of cells. The method uses skeletonization, a technique from morphological image processing to reduce a shape into a series of connected lines. It involves a series of automatic algorithms including image segmentation, boundary smoothing, skeletonization and branch pruning, and takes into account the cell shape changes between successive frames to detect protrusion and retraction activities. In addition, the activities are clustered into different groups, each representing the protruding and retracting history of an individual pseudopod. Conclusions We illustrate the algorithms on movies of chemotaxing Dictyostelium cells and show that our method makes it possible to capture the spatial and temporal dynamics as well as the stochastic features of the pseudopodial behavior. Thus, the method provides a powerful tool for investigating amoeboid motility.

Kinesins are a diverse superfamily of motor proteins that drive organelles and other microtubule-based movements in eukaryotic cells. These motors play important roles in multiple events during both interphase and cell division. Dictyostelium discoideum contains 13 kinesin motors, 12 of which are grouped into nine families, plus one orphan. Functions for 11 of the 13 motors have been previously investigated; we address here the activities of the two remaining kinesins, both isoforms with central motor domains. Kif6 (of the kinesin-13 family) appears to be essential for cell viability. The partial knockdown of Kif6 with RNA interference generates mitotic defects (lagging chromosomes and aberrant spindle assemblies) that are consistent with kinesin-13 disruptions in other organisms. However, the orphan motor Kif9 participates in a completely novel kinesin activity, one that maintains a connection between the microtubule-organizing center (MTOC) and nucleus during interphase. kif9 null cell growth is impaired, and the MTOC appears to disconnect from its normally tight nuclear linkage. Mitotic spindles elongate in a normal fashion in kif9 − cells, but we hypothesize that this kinesin is important for positioning the MTOC into the nuclear envelope during prophase. This function would be significant for the early steps of cell division and also may play a role in regulating centrosome replication.

Background Many cellular processes involve substantial shape changes. Traditional simulations of these cell shape changes require that grids and boundaries be moved as the cell's shape evolves. Here we demonstrate that accurate cell shape changes can be recreated using level set methods (LSM), in which the cellular shape is defined implicitly, thereby eschewing the need for updating boundaries. Results We obtain a viscoelastic model of Dictyostelium cells using micropipette aspiration and show how this viscoelastic model can be incorporated into LSM simulations to recreate the observed protrusion of cells into the micropipette faithfully. We also demonstrate the use of our techniques by simulating the cell shape changes elicited by the chemotactic response to an external chemoattractant gradient. Conclusion Our results provide a simple but effective means of incorporating cellular deformations into mathematical simulations of cell signaling. Such methods will be useful for simulating important cellular events such as chemotaxis and cytokinesis.

Background Cell shape changes during cytokinesis and chemotaxis require regulation of the actin cytoskeletal network. Dynacortin, an actin cross-linking protein, localizes to the cell cortex and contributes to cortical resistance, thereby helping to define the cell shape changes of cytokinesis. Dynacortin also becomes highly enriched in cortical protrusions, which are sites of new actin assembly. Results We studied the effect of dynacortin on cell motility during chemotaxis and on actin dynamics in vivo and in vitro. Dynacortin enriches with the actin, particularly at the leading edge of chemotaxing cells. Cells devoid of dynacortin do not become as polarized as wild-type control cells but move with similar velocities as wild-type cells. In particular, they send out multiple pseudopods that radiate at a broader distribution of angles relative to the chemoattractant gradient. Wild-type cells typically only send out one pseudopod at a time that does not diverge much from 0° on average relative to the gradient. Though dynacortin-deficient cells show normal bulk (whole-cell) actin assembly upon chemoattractant stimulation, dynacortin can promote actin assembly in vitro. By fluorescence spectroscopy, co-sedimentation and transmission electron microscopy, dynacortin acts as an actin scaffolder in which it assembles actin monomers into polymers with a stoichiometry of 1 Dyn2:1 actin under salt conditions that disfavor polymer assembly. Conclusion Dynacortin contributes to cell polarization during chemotaxis. By cross-linking and possibly stabilizing actin polymers, dynacortin also contributes to cortical viscoelasticity, which may be critical for establishing cell polarity. Though not essential for directional sensing or motility, dynacortin is required to establish cell polarity, the third core feature of chemotaxis.

Essential life processes are heavily controlled by a variety of positive and negative feedback systems. Cytokinesis failure, ultimately leading to aneuploidy, is appreciated as an early step in tumor formation in mammals and is deleterious for all cells. Further, the growing list of cancer predisposition mutations includes a number of genes whose proteins control mitosis and/or cytokinesis. Cytokinesis shape control is also an important part of pattern formation and cell-type specialization during multi-cellular development. Inherently mechanical, we hypothesized that mechanosensing and mechanical feedback are fundamental for cytokinesis shape regulation. Using mechanical perturbation, we identified a mechanosensory control system that monitors shape progression during cytokinesis. In this review, we summarize these findings and their implications for cytokinesis regulation and for understanding the cytoskeletal system architecture that governs shape control.

SummaryBecause cell-division failure is deleterious, promoting tumorigenesis in mammals [1], cells utilize numerous mechanisms to control their cell-cycle progression [2–4]. Though cell division is considered a well-ordered sequence of biochemical events [5], cytokinesis, an inherently mechanical process, must also be mechanically controlled to ensure that two equivalent daughter cells are produced with high fidelity. Given that cells respond to their mechanical environment [6, 7], we hypothesized that cells utilize mechanosensing and mechanical feedback to sense and correct shape asymmetries during cytokinesis. Because the mitotic spindle and myosin II are vital to cell division [8, 9], we explored their roles in responding to shape perturbations during cell division. We demonstrate that the contractile proteins myosin II and cortexillin I redistribute in response to intrinsic and externally induced shape asymmetries. In early cytokinesis, mechanical load overrides spindle cues and slows cytokinesis progression while contractile proteins accumulate and correct shape asymmetries. In late cytokinesis, mechanical perturbation also directs contractile proteins but without apparently disrupting cytokinesis. Significantly, this response only occurs during anaphase through cytokinesis, does not require microtubules, and is independent of spindle orientation, but is dependent on myosin II. Our data provide evidence for a mechanosensory system that directs contractile proteins to regulate cell shape during mitosis.

Cell cortices rearrange dynamically to complete cytokinesis, crawlin response to chemoattractant, build tissues, and make neuronal connections. Highly enriched in the cell cortex, actin, myosin II, and actin crosslinkers facilitate cortical movements. Because cortical behavior is the consequence of nanoscale biochemical events, it is essential to probe the cortex at this level. Here, we use high-resolution laser-based particle tracking to examine how myosin II mechanochemistry and dynacortin-mediated actin crosslinking control cortex dynamics in Dictyostelium . Consistent with its low duty ratio, myosin II does not directly drive active bead motility. Instead, myosin II and dynacortin antagonistically regulate other active processes in the living cortex.

Cytokinesis, the fission of a mother cell into two daughter cells, is a simple and dramatic cell shape change. Here, we examine the dynamics of cytokinesis by using a combination of microscopy, dynamic measurements, and genetic analysis. We find that cytokinesis proceeds through a single sequence of shape changes, but the kinetics of the transformation from one shape to another differs dramatically between strains. We interpret the measurements in a simple and quantitative manner by using a previously uncharacterized analytic model. From the analysis, wild-type cytokinesis appears to proceed through an active, extremely regulated process in which globally distributed proteins generate resistive forces that slow the rate of furrow ingression. Finally, we propose that, in addition to myosin II, a Laplace pressure, resulting from material properties and the geometry of the dividing cell, generates force to help drive furrow ingression late in cytokinesis.

During cytokinesis, global and equatorial pathways deform the cell cortex in a stereotypical manner, which leads to daughter cell separation. Equatorial forces are largely generated by myosin-II and the actin crosslinker, cortexillin-I. In contrast, global mechanics are determined by the cortical cytoskeleton, including the actin crosslinker, dynacortin. We used direct morphometric characterization and laser-tracking microrheology to quantify cortical mechanical properties of wild-type and cortexillin-I and dynacortin mutant Dictyostelium cells. Both cortexillin-I and dynacortin influence cytokinesis and interphase cortical viscoelasticity as predicted from genetics and biochemical data using purified dynacortin proteins. Our studies suggest that the regulation of cytokinesis ultimately requires modulation of proteins that control the cortical mechanical properties that establish the force-balance that specifies the shapes of cytokinesis. The combination of genetic, biochemical, and biophysical observations suggests that the cell's cortical mechanical properties control how the cortex is remodeled during cytokinesis.

Cytokinesis is the mechanical process that allows the simplest unit of life, the cell, to divide, propagating itself. To divide, the cell converts chemical energy into mechanical energy to produce force. This process is thought to be active, due in large part to the mechanochemistry of the myosin-II ATPase. The cell's viscoelasticity defines the context and perhaps the magnitude of the forces that are required for cytokinesis. The viscoelasticity may also guide the force-generating apparatus, specifying the cell shape change that results. Genetic, biochemical, and mechanical measurements are providing a quantitative view of how real proteins control this essential life process.

Technical advances are providing new insights into the mechanical properties of cells. Now, by combining genetic and biochemical studies with high-resolution mechanical measurements from atomic force microscopy, the biochemical bases of mechanical processes such as cytokinesis should be discernible.

In this review, we focus on recent discoveries regarding the molecular basis of cleavage furrow positioning and contractile ring assembly and contraction during cytokinesis. However, some of these mechanisms might have different degrees of importance in different organisms. This synthesis attempts to uncover common themes and to reveal potential relationships that might contribute to the biochemical and mechanical aspects of cytokinesis. Because the information about cytokinesis is still fairly rudimentary, our goal is not to present a definitive model but to present testable hypotheses that might lead to a better mechanistic understanding of the process.