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.

Polar relaxation by dynein-mediated removal of cortical myosin II.

Nearly six 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. While this mechanism has remained controversial, recent work has provided evidence for polar relaxation by astral microtubules, although its molecular mechanisms remain elusive. Here, using C.

Rapid production of pure recombinant actin isoforms in .

Actins are major eukaryotic cytoskeletal proteins, and they are involved in 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 for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified by means of an affinity tag introduced in the fusion.

Motor Activity Dependent and Independent Functions of Myosin II Contribute to Actomyosin Ring Assembly and Contraction in Schizosaccharomyces pombe.

Cytokinesis depends on a contractile actomyosin ring in many eukaryotes [1-3]. 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].

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.

Yeast endocytic adaptor AP-2 binds the stress sensor Mid2 and functions in polarized cell responses.

The AP-2 complex is a heterotetrameric endocytic cargo-binding adaptor that facilitates uptake of membrane proteins during mammalian clathrin-mediated endocytosis. While budding yeast has clear homologues of all four AP-2 subunits which form a complex and localize to endocytic sites in vivo, the function of yeast AP-2 has remained enigmatic. Here, we demonstrate that AP-2 is required for hyphal growth in Candida albicans and polarized cell responses in Saccharomyces cerevisiae.

The mating projections of Saccharomyces cerevisiae and Candida albicans show key characteristics of hyphal growth.

Fungi can grow in a variety of growth forms: yeast, pseudohyphae and hyphae. The human fungal pathogen Candida albicans can grow in all three of these forms. In this fungus, hyphal growth is distinguished by the presence of a Spitzenkörper-like structure at the hyphal tip and a band of septin bars around the base of newly evaginated germ tubes.

Hyphal growth in Candida albicans requires the phosphorylation of Sec2 by the Cdc28-Ccn1/Hgc1 kinase.

Polarized growth is a fundamental property of cell growth and development. It requires the delivery of post-Golgi secretory vesicles to the site of polarized growth. This process is mediated by Rab GTPases activated by their guanine exchange factors (GEFs).

The G1 cyclin Cln3 regulates morphogenesis in Candida albicans.

In Saccharomyces cerevisiae, the G1 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 G1 cyclin Cln3 they increased in size but did not bud.