Cleavage-furrow formation without F-actin inChlamydomonas

It is widely believed that cleavage-furrow formation during cytokinesis is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green algaChlamydomonas reinhardtii. We found that although F-actin is associated with the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the largeChlamydomonaschloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cytokinesis itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that microtubules are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.

Cleavage-furrow formation without F-actin in Chlamydomonas

It is widely believed that cleavage-furrow formation during cell division is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green alga Chlamydomonas reinhardtii. We found that although F-actin is concentrated in the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the large Chlamydomonas chloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cell division itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that at least the microtubule plus-ends are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.

Myosin II Tension Sensors Visualize Force Generation within the Actin Cytoskeleton in Living Cells

Type II myosin motors generate cytoskeletal forces that are central to cell division, embryogenesis, muscle contraction, and many other cellular functions. However, at present there is no method that can directly measure the forces generated by myosins in living cells. Here we describe a Förster resonance energy transfer (FRET)-based tension sensor that can measure forces generated by Nonmuscle Myosin IIB (NMIIB) in living cells with piconewton (pN) sensitivity. Fluorescence lifetime imaging microscopy (FLIM)-FRET measurements indicate that the forces generated by NMIIB exhibit significant spatial and temporal heterogeneity, with inferred tensions that vary widely in different regions of the cell. This initial report highlights the potential utility of myosin-based tension sensors in elucidating the roles of cytoskeletal contractility in a wide variety of contexts.

Role of the Hof1–Cyk3 interaction in cleavage-furrow ingression and primary-septum formation during yeast cytokinesis

In Saccharomyces cerevisiae, it is well established that Hof1, Cyk3, and Inn1 contribute to septum formation and cytokinesis. Because hof1∆ and cyk3∆ single mutants have relatively mild defects but hof1∆ cyk3∆ double mutants are nearly dead, it has been hypothesized that these proteins contribute to parallel pathways. However, there is also evidence that they interact physically. In this study, we examined this interaction and its functional significance in detail. Our data indicate that the interaction 1) is mediated by a direct binding of the Hof1 SH3 domain to a proline-rich motif in Cyk3; 2) occurs specifically at the time of cytokinesis but is independent of the (hyper)phosphorylation of both proteins that occurs at about the same time; 3) is dispensable for the normal localization of both proteins; 4) is essential for normal primary-septum formation and a normal rate of cleavage-furrow ingression; and 5) becomes critical for growth when either Inn1 or the type II myosin Myo1 (a key component of the contractile actomyosin ring) is absent. The similarity in phenotype between cyk3∆ mutants and mutants specifically lacking the Hof1–Cyk3 interaction suggests that the interaction is particularly important for Cyk3 function, but it may be important for Hof1 function as well.

Relationships between maximal strength, muscle size, and myosin heavy chain isoform composition and postactivation potentiation

The purpose of this study was to examine the relationships between maximal voluntary postactivation potentiation (PAP) and maximal knee extensor torque, quadriceps cross-sectional area (CSA) and volume, and type II myosin heavy chain (MHC) isoform percentage in human skeletal muscle. Thirteen resistance-trained men completed a test protocol consisting of 2 isokinetic knee extensions at 180°·s–1 performed before and 1, 4, 7, and 10 min after the completion of 4 maximal knee extensions at 60°·s–1 (i.e., a conditioning activity (CA)). Magnetic resonance imaging and muscle microbiopsy procedures were completed on separate days to assess quadriceps CSA and volume and MHC isoform content. Maximal voluntary PAP response was assessed as the ratio of the highest knee extensor torques measured before and after the CA. There were large to very large correlations between maximal voluntary PAP response and maximal knee extensor torque (r = 0.62) and quadriceps CSA (r = 0.68) and volume (r = 0.63). Nonetheless, these correlations were not statistically significant after adjusting for the influence of type II MHC percentage using partial correlation analysis. By contrast, the strongest correlation was observed for type II MHC percentage (r = 0.77), and this correlation remained significant after adjusting for the other variables. Maximal voluntary PAP response is strongly correlated with maximal knee extensor torque and quadriceps CSA and volume, but is mostly clearly associated with the type II myosin isoform percentage in human skeletal muscle.

Dephosphorylation of Iqg1 by Cdc14 regulates cytokinesis in budding yeast

Cytokinesis separates cells by contraction of a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential protein in Saccharomyces cerevisiae required for assembly and contraction of the actomyosin ring. Localization of F-actin to the ring occurs only after anaphase and is mediated by the calponin homology domain (CHD) of Iqg1, but the regulatory mechanisms that temporally restrict actin ring assembly are not well defined. We tested the hypothesis that dephosphorylation of four perfect cyclin-dependent kinase (Cdk) sites flanking the CHD promotes actin ring formation, using site-specific alanine mutants. Cells expressing the nonphosphorylatable iqg1-4A allele formed actin rings before anaphase and exhibited defects in myosin contraction and cytokinesis. The Cdc14 phosphatase is required for normal cytokinesis and acts on specific Cdk phosphorylation sites. Overexpression of Cdc14 resulted in premature actin ring assembly, whereas inhibition of Cdc14 function prevented actin ring formation. Cdc14 associated with Iqg1, dependent on several CHD-flanking Cdk sites, and efficiently dephosphorylated these sites in vitro. Of importance, the iqg1-4A mutant rescued the inability of cdc14-1 cells to form actin rings. Our data support a model in which dephosphorylation of Cdk sites around the Iqg1 CHD by Cdc14 is both necessary and sufficient to promote actin ring formation. Temporal control of actin ring assembly by Cdk and Cdc14 may help to ensure that cytokinesis onset occurs after nuclear division is complete.

Aim44p regulates phosphorylation of Hof1p to promote contractile ring closure during cytokinesis in budding yeast

Whereas actomyosin and septin ring organization and function in cytokinesis are thoroughly described, little is known regarding the mechanisms by which the actomyosin ring interacts with septins and associated proteins to coordinate cell division. Here we show that the protein product of YPL158C, Aim44p, undergoes septin-dependent recruitment to the site of cell division. Aim44p colocalizes with Myo1p, the type II myosin of the contractile ring, throughout most of the cell cycle. The Aim44p ring does not contract when the actomyosin ring closes. Instead, it forms a double ring that associates with septin rings on mother and daughter cells after cell separation. Deletion of AIM44 results in defects in contractile ring closure. Aim44p coimmunoprecipitates with Hof1p, a conserved F-BAR protein that binds both septins and type II myosins and promotes contractile ring closure. Deletion of AIM44 results in a delay in Hof1p phosphorylation and altered Hof1p localization. Finally, overexpression of Dbf2p, a kinase that phosphorylates Hof1p and is required for relocalization of Hof1p from septin rings to the contractile ring and for Hof1p-triggered contractile ring closure, rescues the cytokinesis defect observed in aim44∆ cells. Our studies reveal a novel role for Aim44p in regulating contractile ring closure through effects on Hof1p.