Contractile Research Articles

A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di‐phosphorylated myosin light chain (ppMLC)‐driven contraction of actomyosin‐based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC‐triggered system at TJ‐associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule‐associated proteins in the AJC‐enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ‐, but not at AJ‐, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ‐associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule‐facilitated manner. Our results uncovered a hitherto unknown microtubule‐LUZP1 association at TJ‐associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction.

Time-varying mobility and turnover of actomyosin ring components during cytokinesis in Schizosaccharomyces pombe.

Cytokinesis in many eukaryotes is dependent on a contractile actomyosin ring (AMR), composed of F-actin, myosin II, and other actin and myosin II regulators. Through fluorescence recovery after photobleaching experiments, many components of the AMR have been shown to be mobile and to undergo constant exchange with the cytosolic pools. However, how the mobility of its components changes at distinct stages of mitosis and cytokinesis has not been addressed. Here, we describe the mobility of eight Schizosaccharomyces pombe AMR proteins at different stages of mitosis and cytokinesis using an approach we have developed. We identified three classes of proteins, which showed 1) high (Ain1, Myo2, Myo51), 2) low (Rng2, Mid1, Myp2, Cdc12), and 3) cell cycle–dependent (Cdc15) mobile fractions. We observed that the F-BAR protein Cdc15 undergoes a 20–30% reduction in its mobile fraction after spindle breakdown and initiation of AMR contraction. Moreover, our data indicate that this change in Cdc15 mobility is dependent on the septation initiation network (SIN). Our work offers a novel strategy for estimating cell cycle–dependent mobile protein fractions in cellular structures and provides a valuable dataset, that is of interest to researchers working on cytokinesis.

The kinetic landscape and interplay of protein networks in cytokinesis

SummaryCytokinesis is executed by protein networks organized into functional modules. Individual proteins within each module have been characterized to various degrees. However, the collective behavior and interplay of the modules remain poorly understood. In this study, we conducted quantitative time-lapse imaging to analyze the accumulation kinetics of more than 20 proteins from different modules of cytokinesis in budding yeast. This analysis has led to a comprehensive picture of the kinetic landscape of cytokinesis, from actomyosin ring (AMR) assembly to cell separation. It revealed that the AMR undergoes biphasic constriction and that the switch between the constriction phases is likely triggered by AMR maturation and primary septum formation. This analysis also provided further insights into the functions of actin filaments and the transglutaminase-like protein Cyk3 in cytokinesis and, in addition, defined Kre6 as the likely enzyme that catalyzes β-1,6-glucan synthesis to drive cell wall maturation during cell growth and division.

Autocrine insulin pathway signaling regulates actin dynamics in cell wound repair.

Cells are exposed to frequent mechanical and/or chemical stressors that can compromise the integrity of the plasma membrane and underlying cortical cytoskeleton. The molecular mechanisms driving the immediate repair response launched to restore the cell cortex and circumvent cell death are largely unknown. Using microarrays and drug-inhibition studies to assess gene expression, we find that initiation of cell wound repair in the Drosophila model is dependent on translation, whereas transcription is required for subsequent steps. We identified 253 genes whose expression is up-regulated (80) or down-regulated (173) in response to laser wounding. A subset of these genes were validated using RNAi knockdowns and exhibit aberrant actomyosin ring assembly and/or actin remodeling defects. Strikingly, we find that the canonical insulin signaling pathway controls actin dynamics through the actin regulators Girdin and Chickadee (profilin), and its disruption leads to abnormal wound repair. Our results provide new insight for understanding how cell wound repair proceeds in healthy individuals and those with diseases involving wound healing deficiencies.

The phosphatase inhibitor Sds23 promotes symmetric spindle positioning in fission yeast.

A hallmark of cell division in eukaryotic cells is the formation and elongation of a microtubule (MT)-based mitotic spindle. Proper positioning of the spindle is critical to ensure equal segregation of the genetic material to the resulting daughter cells. Both the timing of spindle elongation and constriction of the actomyosin contractile ring must be precisely coordinated to prevent missegregation or damage to the genetic material during cellular division. Here, we show that Sds23, an inhibitor of protein phosphatases, contributes to proper positioning of elongating spindles in fission yeast cells. We found that sds23∆ mutant cells exhibit asymmetric spindles that initially elongate asymmetrically toward one end of the dividing cell. Spindle asymmetry in sds23∆ cells results from a defect that is distinct from previously identified mechanisms, including MT protrusions and enlarged vacuoles. Combined with our previous work, this study demonstrates that Sds23, an inhibitor of PP2A-family protein phosphatases, promotes proper positioning of both the bipolar spindle and cytokinetic ring during fission yeast cell division. These two steps ensure the overall symmetry and fidelity of the cell division process.

Assembly properties of bacterial tubulin homolog FtsZ regulated by the positive regulator protein ZipA and ZapA from Pseudomonas aeruginosa

Bacterial tubulin homolog FtsZ self-assembles into dynamic protofilaments, which forms the scaffold for the contractile ring (Z-ring) to achieve bacterial cell division. Here, we study the biochemical properties of FtsZ from Pseudomonas aeruginosa (PaFtsZ) and the effects of its two positive regulator proteins, ZipA and ZapA. Similar to Escherichia coli FtsZ, PaFtsZ had a strong GTPase activity, ~ 7.8 GTP min-1 FtsZ-1 at pH 7.5, and assembled into mainly short single filaments in vitro. However, PaFtsZ protofilaments were mixtures of straight and “intermediate-curved” (100–300 nm diameter) in pH 7.5 solution and formed some bundles in pH 6.5 solution. The effects of ZipA on PaFtsZ assembly varied with pH. In pH 6.5 buffer ZipA induced PaFtsZ to form large bundles. In pH 7.5 buffer PaFtsZ-ZipA protofilaments were not bundled, but ZipA enhanced PaFtsZ assembly and promoted more curved filaments. Comparable to ZapA from other bacterial species, ZapA from P. aeruginosa induced PaFtsZ protofilaments to associate into long straight loose bundles and/or sheets at both pH 6.5 and pH 7.5, which had little effect on the GTPase activity of PaFtsZ. These results provide us further information that ZipA functions as an enhancer of FtsZ curved filaments, while ZapA works as a stabilizer of FtsZ straight filaments.

액틴 필라멘트의 강직도가 네트워크의 수축에 미치는 영향

액틴 필라멘트의 강직도가 네트워크의 수축에 미치는 영향

A new role of glutathione peroxidase 4 during human erythroblast enucleation

AbstractThe selenoprotein glutathione peroxidase 4 (GPX4), the only member of the glutathione peroxidase family able to directly reduce cell membrane–oxidized fatty acids and cholesterol, was recently identified as the central regulator of ferroptosis. GPX4 knockdown in mouse hematopoietic cells leads to hemolytic anemia and to increased spleen erythroid progenitor death. The role of GPX4 during human erythropoiesis is unknown. Using in vitro erythroid differentiation, we show here that GPX4-irreversible inhibition by 1S,3R-RSL3 (RSL3) and its short hairpin RNA–mediated knockdown strongly impaired enucleation in a ferroptosis-independent manner not restored by tocopherol or iron chelators. During enucleation, GPX4 localized with lipid rafts at the cleavage furrows between reticulocytes and pyrenocytes. Its inhibition impacted enucleation after nuclear condensation and polarization and was associated with a defect in lipid raft clustering (cholera toxin staining) and myosin-regulatory light-chain phosphorylation. Because selenoprotein translation and cholesterol synthesis share a common precursor, we investigated whether the enucleation defect could represent a compensatory mechanism favoring GPX4 synthesis at the expense of cholesterol, known to be abundant in lipid rafts. Lipidomics and filipin staining failed to show any quantitative difference in cholesterol content after RSL3 exposure. However, addition of cholesterol increased cholera toxin staining and myosin-regulatory light-chain phosphorylation, and improved enucleation despite GPX4 knockdown. In summary, we identified GPX4 as a new actor of human erythroid enucleation, independent of its function in ferroptosis control. We described its involvement in lipid raft organization required for contractile ring assembly and cytokinesis, leading in fine to nucleus extrusion.

Comparative Analysis of the Roles of Non-muscle Myosin-IIs in Cytokinesis in Budding Yeast, Fission Yeast, and Mammalian Cells.

The contractile ring, which plays critical roles in cytokinesis in fungal and animal cells, has fascinated biologists for decades. However, the basic question of how the non-muscle myosin-II and actin filaments are assembled into a ring structure to drive cytokinesis remains poorly understood. It is even more mysterious why and how the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and humans construct the ring structure with one, two, and three myosin-II isoforms, respectively. Here, we provide a comparative analysis of the roles of the non-muscle myosin-IIs in cytokinesis in these three model systems, with the goal of defining the common and unique features and highlighting the major questions regarding this family of proteins.

Rac and Arp2/3-Nucleated Actin Networks Antagonize Rho During Mitotic and Meiotic Cleavages.

In motile cells, the activities of the different Rho family GTPases are spatially segregated within the cell, and during cytokinesis there is evidence that this may also be the case. But while Rho’s role as the central organizer for contractile ring assembly is well established, the role of Rac and the branched actin networks it promotes is less well understood. To characterize the contributions of these proteins during cytokinesis, we manipulated Rac and Arp2/3 activity during mitosis and meiosis in sea urchin embryos and sea star oocytes. While neither Rac nor Arp2/3 were essential for early embryonic divisions, loss of either Rac or Arp2/3 activity resulted in polar body defects. Expression of activated Rac resulted in cytokinesis failure as early as the first division, and in oocytes, activated Rac suppressed both the Rho wave that traverses the oocyte prior to polar body extrusion as well as polar body formation itself. However, the inhibitory effect of Rac on cytokinesis, polar body formation and the Rho wave could be suppressed by effector-binding mutations or direct inhibition of Arp2/3. Together, these results suggest that Rac- and Arp2/3 mediated actin networks may directly antagonize Rho signaling, thus providing a potential mechanism to explain why Arp2/3-nucleated branched actin networks must be suppressed at the cell equator for successful cytokinesis.

The Effect of Rebamipide on the Regulation of Intestinal Contractility

The Effect of Rebamipide on the Regulation of Intestinal Contractility

Calcium spikes accompany cleavage furrow ingression and cell separation during fission yeast cytokinesis.

The role of calcium signaling in cytokinesis has long remained ambiguous. Past studies of embryonic cell division discovered that calcium concentration increases transiently at the division plane just before cleavage furrow ingression, suggesting that these calcium transients could trigger contractile ring constriction. However, such calcium transients have only been found in animal embryos and their function remains controversial. We explored cytokinetic calcium transients in the fission yeast Schizosaccharomyces pombe by adopting GCaMP, a genetically encoded calcium indicator, to determine the intracellular calcium level of this model organism. We validated GCaMP as a highly sensitive calcium reporter in fission yeast, allowing us to capture calcium transients triggered by osmotic shocks. We identified a correlation between the intracellular calcium level and cell division, consistent with the existence of calcium transients during cytokinesis. Using time-lapse microscopy and quantitative image analysis, we discovered calcium spikes both at the start of cleavage furrow ingression and the end of cell separation. Inhibition of these calcium spikes slowed the furrow ingression and led to frequent lysis of daughter cells. We conclude that like the larger animal embryos, fission yeast triggers calcium transients that may play an important role in cytokinesis (197).

A novel coordinated function of Myosin II with GOLPH3 controls centralspindlin localization during cytokinesis in Drosophila.

In animal cell cytokinesis, interaction of non-muscle myosin II (NMII) with F-actin provides the dominant force for pinching the mother cell into two daughters. Here we demonstrate that celibe (cbe) is a missense allele of zipper, which encodes the Drosophila Myosin heavy chain. Mutation of cbe impairs binding of Zipper protein to the regulatory light chain Spaghetti squash (Sqh). In dividing spermatocytes from cbe males, Sqh fails to concentrate at the equatorial cortex, resulting in thin actomyosin rings that are unable to constrict. We show that cbe mutation impairs localization of the phosphatidylinositol 4-phosphate [PI(4)P]-binding protein Golgi phosphoprotein 3 (GOLPH3, also known as Sauron) and maintenance of centralspindlin at the cell equator of telophase cells. Our results further demonstrate that GOLPH3 protein associates with Sqh and directly binds the centralspindlin subunit Pavarotti. We propose that during cytokinesis, the reciprocal dependence between Myosin and PI(4)P-GOLPH3 regulates centralspindlin stabilization at the invaginating plasma membrane and contractile ring assembly.

Alteration in the Global and Regional Left Ventricular and Right Ventricular Myocardial Strain Patterns in Patients with Inferior ST-Elevation Myocardial Infarction Early and Late after Percutaneous Coronary Intervention

Background: Precise evaluation of regional or global heart contractility is very important for the diagnosis, decision of treatment options, and prognosis of clinical outcomes in the Myocardial Ischemia (MI) and infarction. Aims: Evaluation of the regional or global distortion of Left Ventricle (LV) and Right Ventricle (RV) in cases with Inferior Wall Myocardial Ischemia (IWMI) early and late afterward percutaneous coronary treatment. Materials and Methods: This prospective study was conducted at Monofia University Hospital during the period from November 2017 till December 2019. The study included patients who presented to the cardiology department with inferior ST Segment Elevation MI (STEMI). Only 40 patients fulfilled the criteria of inclusion and were classified to 2 groups; Group I: include cases suffering from inferior STEMI. Group II (control): 20 healthy volunteer subjects. Results: This study showed that LV global strain were reduced with highly significant value in cases suffering from IWMI prior to Percutaneous Coronary Intervention (PCI) than in controls (-11.10±1.75 vs -19.84±1.37) P value <0.001 respectively While LV global strain was significantly improved in cases with post-PCI than pre-PCI (-15.86±1.50 vs -11.10 ±1.75) P value < 0.001 respectively. Conclusion: Reperfusion by PCI outcomes in significant recovery of LV and RV global strains, which are at most exist in the infarct and adjacent myocardium of apical and middle segments.

Cell cycle-dependent phosphorylation of IQGAP is involved in assembly and stability of the contractile ring in fission yeast

Cytokinesis is the final step in cell division and is driven by the constriction of the medial actomyosin-based contractile ring (CR) in many eukaryotic cells. In the fission yeast Schizosaccharomyces pombe, the IQGAP-like protein Rng2 is required for assembly and constriction of the CR, and specifically interacts with actin filaments (F-actin) in the CR after anaphase. However, the mechanism that timely activates Rng2 has not yet been elucidated. We herein tested the hypothesis that the cytokinetic function of Rng2 is regulated by phosphorylation by examining phenotypes of a series of non-phosphorylatable and phosphomimetic rng2 mutant strains. In phosphomimetic mutant cells, F-actin in the CR was unstable. Genetic analyses indicated that phosphorylated Rng2 was involved in CR assembly in cooperation with myosin-II, whereas the phosphomimetic mutation attenuated the localization of Rng2 to CR F-actin. The present results suggest that Rng2 is phosphorylated during CR assembly and then dephosphorylated, which enhances the interaction between Rng2 and CR F-actin to stabilize the ring, thereby ensuring secure cytokinesis.

A novel checkpoint pathway controls actomyosin ring constriction trigger in fission yeast.

In fission yeast, the septation initiation network (SIN) ensures temporal coordination between actomyosin ring (CAR) constriction with membrane ingression and septum synthesis. However, questions remain about CAR regulation under stress conditions. We show that Rgf1p (Rho1p GEF), participates in a delay of cytokinesis under cell wall stress (blankophor, BP). BP did not interfere with CAR assembly or the rate of CAR constriction, but did delay the onset of constriction in the wild type cells but not in the rgf1Δ cells. This delay was also abolished in the absence of Pmk1p, the MAPK of the cell integrity pathway (CIP), leading to premature abscission and a multi-septated phenotype. Moreover, cytokinesis delay correlates with maintained SIN signaling and depends on the SIN to be achieved. Thus, we propose that the CIP participates in a checkpoint, capable of triggering a CAR constriction delay through the SIN pathway to ensure that cytokinesis terminates successfully.

The Kinetic Landscape and Interplay of Protein Networks in Cytokinesis

Cytokinesis is a complex process accomplished by strict spatiotemporal coordination of distinct functional modules, including assembly and constriction of a cortical actomyosin ring (AMR), targeted vesicle fusion, and localized extracellular matrix remodeling. Each module consists of multiple proteins whose individual roles have been characterized in different model systems. However, the collective behaviors and interplay of the modules under normal and stressed conditions remain poorly understood. In this study, we conducted quantitative time-lapse imaging to analyze the accumulation kinetics of more than 20 proteins from different modules of cytokinesis in budding yeast. This analysis has provided the first picture of the kinetic landscape of cytokinesis, from AMR assembly to cell separation or abscission. This analysis has also revealed that the AMR undergoes biphasic constriction, akin to other model systems, and the switch between the constriction phases is likely triggered by the AMR maturation and primary septum formation. This microscopy-based kinetic analysis, when combined with chemical or genetic perturbations, has provided us with significant mechanistic insights into the functions of actin filaments and the transglutaminase-like protein Cyk3 in cytokinesis. Importantly, using the kinetic signature, i.e. the peaking time of each cytokinetic module, as a predictive parameter, coupled with functional analysis, we have found that the type-II membrane protein Kre6 catalyzes β-1,6-glucan synthesis and may also coordinate its function with the Fks1-mediated β-1,3-glucan synthesis to drive cell wall maturation before cell separation.

Animal Cell Cytokinesis: The Rho-Dependent Actomyosin-Anilloseptin Contractile Ring as a Membrane Microdomain Gathering, Compressing, and Sorting Machine.

Cytokinesis is the last step of cell division that partitions the cellular organelles and cytoplasm of one cell into two. In animal cells, cytokinesis requires Rho-GTPase-dependent assembly of F-actin and myosin II (actomyosin) to form an equatorial contractile ring (CR) that bisects the cell. Despite 50 years of research, the precise mechanisms of CR assembly, tension generation and closure remain elusive. This hypothesis article considers a holistic view of the CR that, in addition to actomyosin, includes another Rho-dependent cytoskeletal sub-network containing the scaffold protein, Anillin, and septin filaments (collectively termed anillo-septin). We synthesize evidence from our prior work in Drosophila S2 cells that actomyosin and anillo-septin form separable networks that are independently anchored to the plasma membrane. This latter realization leads to a simple conceptual model in which CR assembly and closure depend upon the micro-management of the membrane microdomains to which actomyosin and anillo-septin sub-networks are attached. During CR assembly, actomyosin contractility gathers and compresses its underlying membrane microdomain attachment sites. These microdomains resist this compression, which builds tension. During CR closure, membrane microdomains are transferred from the actomyosin sub-network to the anillo-septin sub-network, with which they flow out of the CR as it advances. This relative outflow of membrane microdomains regulates tension, reduces the circumference of the CR and promotes actomyosin disassembly all at the same time. According to this hypothesis, the metazoan CR can be viewed as a membrane microdomain gathering, compressing and sorting machine that intrinsically buffers its own tension through coordination of actomyosin contractility and anillo-septin-membrane relative outflow, all controlled by Rho. Central to this model is the abandonment of the dogmatic view that the plasma membrane is always readily deformable by the underlying cytoskeleton. Rather, the membrane resists compression to build tension. The notion that the CR might generate tension through resistance to compression of its own membrane microdomain attachment sites, can account for numerous otherwise puzzling observations and warrants further investigation using multiple systems and methods.

C. elegans CLASP/CLS-2 negatively regulates membrane ingression throughout the oocyte cortex and is required for polar body extrusion.

The requirements for oocyte meiotic cytokinesis during polar body extrusion are not well understood. In particular, the relationship between the oocyte meiotic spindle and polar body contractile ring dynamics remains largely unknown. We have used live cell imaging and spindle assembly defective mutants lacking the function of CLASP/CLS-2, kinesin-12/KLP-18, or katanin/MEI-1 to investigate the relationship between meiotic spindle structure and polar body extrusion in C. elegans oocytes. We show that spindle bipolarity and chromosome segregation are not required for polar body contractile ring formation and chromosome extrusion in klp-18 mutants. In contrast, oocytes with similarly severe spindle assembly defects due to loss of CLS-2 or MEI-1 have penetrant and distinct polar body extrusion defects: CLS-2 is required early for contractile ring assembly or stability, while MEI-1 is required later for contractile ring constriction. We also show that CLS-2 both negatively regulates membrane ingression throughout the oocyte cortex during meiosis I, and influences the dynamics of the central spindle-associated proteins Aurora B/AIR-2 and MgcRacGAP/CYK-4. We suggest that proper regulation by CLS-2 of both oocyte cortical stiffness and central spindle protein dynamics may influence contractile ring assembly during polar body extrusion in C. elegans oocytes.

Impairment of cytokinesis by cancer-associated DAPK3 mutations

Death-associated protein kinase 3 (DAPK3), a member of the DAPK family, contributes to cytokinesis by phosphorylating myosin II regulatory light chain (MRLC). Missense mutations in DAPK3, T112M, D161N, and P216S, were observed in the lung, colon, and cervical cancers, respectively, but the effects of these mutations on cytokinesis remain unclear. Here, we show that cells expressing EGFP-DAPK3-T112M, -D161N, or -P216S exhibited reduced rates of cytokinesis, with an increased ratio of multinucleated cells. In addition, these cells exhibited reduced levels of phosphorylated MRLC at the contractile ring. Collectively, our data demonstrates that cancer-associated DAPK3 mutations impair cytokinesis by reducing phosphorylated MRLC.