Polar relaxation by dynein-mediated removal of cortical myosin II

Nearly 6 decades ago, Lewis Wolpert proposed the relaxation of the polar cell cortex by the radial arrays of astral microtubules as a mechanism for cleavage furrow induction (White and Borisy, 1983; Wolpert, 1960). While this mechanism has remained controversial (Rappaport, 1996), recent work has provided evidence for polar relaxation by astral microtubules (Chen et al., 2008; Dechant and Glotzer, 2003; Foe and Dassow, 2008; Murthy and Wadsworth, 2008; Werner et al., 2007), although its molecular mechanisms remain elusive. Here, using C. elegans embryos, we show that polar relaxation is achieved through dynein-mediated removal of myosin II from the polar cortexes. Mutants that position centrosomes closer to the polar cortex accelerated furrow induction whereas suppression of dynein activity delayed furrowing. We provide evidence that dynein-mediated removal of myosin II from the polar cortexes triggers cortical flow towards the cell equator, which induces the assembly of the actomyosin contractile ring. These studies for the first time provide a molecular basis for the aster-dependent polar relaxation, which works in parallel with equatorial stimulation to promote robust cytokinesis

Apm4, the mu subunit of yeast AP-2 interacts with Pkc1, and mutation of the Pkc1 consensus phosphorylation site Thr176 inhibits AP-2 recruitment to endocytic sites.

The AP-2 endocytic adaptor has been extensively characterized in mammalian cells and is considered to play a role both in cargo binding and in formation of endocytic sites. However, despite our detailed knowledge of mechanistic aspects of endocytic complex assembly and disassembly in the model organism Saccharomyces cerevisiae, no function of AP-2 had been described in wild-type yeast under normal growth conditions. A recent study however revealed that disruption of the complex caused by deletion of the gene encoding its mu subunit (APM4) caused defects in cell polarity such that responses to pheromone, nutritional status and cell wall damage were affected. Furthermore, a homozygous deletion of the mu subunit gene in Candida albicans affected its ability to grow hyphae. Direct binding to the yeast cell wall stress sensor Mid2 was detected, and in an apm4 deletion strain Mid2 showed reduced re-localization to the mother bud neck region following cell wall damage with calcofluor or to the mating projection tip. Here we demonstrate an interaction between Apm4 and the yeast cell wall integrity pathway component Pkc1 and show that mutation of the predicted Pkc1 site in the Apm4 hinge region affects recruitment of the AP-2 complex to endocytic sites

Motor activity dependent and independent functions for Myosin II in cytokinesis contribute to actomyosin ring assembly and contraction in Schizosaccharomyces pombe

Cytokinesis depends on a contractile actomyosin ring in many eukaryotes [1, 2, 3]. Myosin II is a key component of the actomyosin ring, although whether it functions as a motor or as an actin cross-linker to exert its essential role is disputed [1, 4, 5]. In Schizosaccharomyces pombe, the myo2-E1 mutation affects the upper 50 kDa sub-domain of the myosin II heavy chain, and cells carrying this lethal mutation are defective in actomyosin ring assembly at the non-permissive temperature [6, 7]. myo2-E1 also affects actomyosin ring contraction when rings isolated from permissive temperature-grown cells are incubated with ATP [8]. Here we report isolation of a compensatory suppressor mutation in the lower 50 kDa sub-domain (myo2-E1-Sup1) that reverses the inability of myo2-E1 to form colonies at the restrictive temperature. myo2-E1-Sup1 is capable of assembling normal actomyosin rings, although rings isolated from myo2-E1-Sup1 are defective in ATP-dependent contraction in vitro. Furthermore, the product of myo2-E1-Sup1 does not translocate actin filaments in motility assays in vitro. Superimposition of myo2-E1 and myo2-E1-Sup1 on available rigor and blebbistatin-bound myosin II structures suggests that myo2-E1-Sup1 may represent a novel actin translocation-defective allele. Actomyosin ring contraction and viability of myo2-E1-Sup1 cells depend on the late cytokinetic S. pombe myosin II isoform, Myp2p, a non-essential protein that is normally dispensable for actomyosin ring assembly and contraction. Our work reveals that Myo2p may function in two different and essential modes during cytokinesis: a motor activity-independent form that can promote actomyosin ring assembly and a motor activity-dependent form that supports ring contraction

Rapid production of pure recombinant actin isoforms in Pichia pastoris

Actins are major eukaryotic cytoskeletal proteins, which perform many important cell functions, including cell division, cell polarity, wound healing, and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively presently for biochemical studies of actin cytoskeleton from other organisms / cell types. Here we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris. Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified using an affinity tag introduced in the fusion. Following cleavage of thymosin β4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from S. cerevisiae, S. pombe, and the β- and γ- isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate actin dendritic networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton

Molecular biology of morphogenesis in Candida albicans : Cln3 has a role in the regulation of the cell cycle and hyphal growth, and Nap1 is required for septin ring fomation only in hyphae

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The G(1) Cyclin Cln3 Regulates Morphogenesis in Candida albicans

In Saccharomyces cerevisiae, the G(1) cyclin Cln3 initiates the Start of a mitotic cell cycle in response to size and nutrient inputs. Loss of Cln3 delays but does not prevent Start, due to the eventual Cln3-independent transcription of CLN1 and CLN2. When unbudded cells of the human pathogen Candida albicans were depleted of the G(1) cyclin Cln3 they increased in size but did not bud. Thus, unlike S. cerevisiae, Cln3 is essential for budding in C. albicans. However, eventually the large unbudded cells spontaneously produced filamentous forms. The morphology was growth medium dependent; on nutritionally poor medium the polarized outgrowths fulfilled the formal criteria for true hyphae. This state is stable, and continued growth leads to a hyphal mycelium, which invades the agar substratum. Interestingly, it is also required for normal hyphal development, as Cln3-depleted cells develop morphological abnormalities if challenged with hyphal inducing signals such as serum or neutral pH. Taken together, these results show that, in C. albicans, Cln3 has assumed a critical role in coordinating mitotic cell division with differentiation

mNG-tagged fusion proteins and nanobodies to visualize tropomyosins in yeast and mammalian cells

Tropomyosins are structurally conserved α-helical coiled-coil proteins that bind along the length of filamentous actin (F-actin) in fungi and animals. Tropomyosins play essential roles in the stability of actin filaments and in regulating myosin II contractility. Despite the crucial role of tropomyosin in actin cytoskeletal regulation, in vivo investigations of tropomyosin are limited, mainly due to the suboptimal live-cell imaging tools currently available. Here, we report on an mNeonGreen (mNG)-tagged tropomyosin, with native promoter and linker length configuration, that clearly reports tropomyosin dynamics in Schizosaccharomyces pombe (Cdc8), Schizosaccharomyces japonicus (Cdc8) and Saccharomyces cerevisiae (Tpm1 and Tpm2). We also describe a fluorescent probe to visualize mammalian tropomyosin (TPM2 isoform). Finally, we generated a camelid nanobody against S. pombe Cdc8, which mimics the localization of mNG–Cdc8 in vivo. Using these tools, we report the presence of tropomyosin in previously unappreciated patch-like structures in fission and budding yeasts, show flow of tropomyosin (F-actin) cables to the cytokinetic actomyosin ring and identify rearrangements of the actin cytoskeleton during mating. These powerful tools and strategies will aid better analyses of tropomyosin and F-actin cables in vivo