Sathish Thiyagarajan

The fission yeast cytokinetic contractile ring regulates septum shape and closure

ABSTRACT During cytokinesis, fission yeast and other fungi and bacteria grow a septum that divides the cell in two. In fission yeast closure of the circular septum hole by the β-glucan synthases (Bgs) and other glucan synthases in the plasma membrane is tightly coupled to constriction of an actomyosin contractile ring attached to the membrane. It is unknown how septum growth is coordinated over scales of several microns to maintain septum circularity. Here, we documented the shapes of ingrowing septum edges by measuring the roughness of the edges, a measure of the deviation from circularity. The roughness was small, with spatial correlations indicative of spatially coordinated growth. We hypothesized that Bgs-mediated septum growth is mechanosensitive and coupled to contractile ring tension. A mathematical model showed that ring tension then generates almost circular septum edges by adjusting growth rates in a curvature-dependent fashion. The model reproduced experimental roughness statistics and showed that septum synthesis sets the mean closure rate. Our results suggest that the fission yeast cytokinetic ring tension does not set the constriction rate but regulates septum closure by suppressing roughness produced by inherently stochastic molecular growth processes. Summary: During cytokinesis in fission yeast the tension of the actomyosin contractile ring regulates septum closure by suppressing roughness of the septum edge, while septum growth sets the constriction rate.

Entropic forces drive self-organization and membrane fusion by SNARE proteins

Significance Secretion of neurotransmitters and hormones depends on membrane fusion, accomplished by a cellular fusion machinery whose core components are SNARE proteins. Several vesicle-associated v-SNAREs form SNAREpin complexes with target membrane-associated t-SNAREs, but how they cooperate to catalyze fusion is unknown. Using highly coarse-grained simulations, we found that steric interactions spontaneously organized SNAREpins into circular clusters, expanded the clusters, and forced membranes together. These are entropic effects, reminiscent of cooperative transitions in solutions of rod-like molecules. The rate of fusion was controlled by the number of SNAREs, because more SNAREs generated greater entropic forces. Given ∼70 v-SNAREs available per synaptic vesicle, entropic cooperativity among SNAREpins may underlie the sub-millisecond timescale of neurotransmitter release. SNARE proteins are the core of the cell’s fusion machinery and mediate virtually all known intracellular membrane fusion reactions on which exocytosis and trafficking depend. Fusion is catalyzed when vesicle-associated v-SNAREs form trans-SNARE complexes (“SNAREpins”) with target membrane-associated t-SNAREs, a zippering-like process releasing ∼65 kT per SNAREpin. Fusion requires several SNAREpins, but how they cooperate is unknown and reports of the number required vary widely. To capture the collective behavior on the long timescales of fusion, we developed a highly coarse-grained model that retains key biophysical SNARE properties such as the zippering energy landscape and the surface charge distribution. In simulations the ∼65-kT zippering energy was almost entirely dissipated, with fully assembled SNARE motifs but uncomplexed linker domains. The SNAREpins self-organized into a circular cluster at the fusion site, driven by entropic forces that originate in steric–electrostatic interactions among SNAREpins and membranes. Cooperative entropic forces expanded the cluster and pulled the membranes together at the center point with high force. We find that there is no critical number of SNAREs required for fusion, but instead the fusion rate increases rapidly with the number of SNAREpins due to increasing entropic forces. We hypothesize that this principle finds physiological use to boost fusion rates to meet the demanding timescales of neurotransmission, exploiting the large number of v-SNAREs available in synaptic vesicles. Once in an unfettered cluster, we estimate ≥15 SNAREpins are required for fusion within the ∼1-ms timescale of neurotransmitter release.

Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs). Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx) and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs) of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores in a biochemically defined setting which have recently become available. Finally, computer simulations are valuable mechanistic tools because they have the power to access small length scales and very short times that are experimentally inaccessible.

A node organization in the actomyosin contractile ring generates tension and aids stability

Recent experiments showed that key cytokinetic ring proteins organize into membrane-anchored complexes called nodes in the fission yeast cytokinetic ring. A mathematical model based on this organization showed that ring tension arises from myosins pulling anchored actin filaments, and component turnover and anchoring ensure structural stability.

Interdependence of Ring Constriction and Septation in Fission Yeast Cytokinesis

Cytokinesis concludes the cell cycle and entails physical division of the cytoplasm into two parts. In animals and fungi, cytokinesis involves constriction of an actomyosin contractile ring coupled to other simultaneous processes but how these processes coordinate with the ring is not established. In fission yeast, constriction is tightly coupled to septation, the growth of new cell wall in the wake of the constricting ring. It is unknown whether ring closure is driven by septation or the ring itself. Here we developed a model of cytokinesis in fission yeast that describes the coupling of the tensile contractile ring to the growth of primary septum by Bgs1p and other proteins that synthesize septum material. The model hypothesizes that ring tension influences the local rate of septum growth through the effect of radial ring-generated forces on Bgs1p or other motors. We used our model to calculate the rate of inward growth of the septum and the evolution of the shape of the inner septum boundary during ring closure. Our model results are consistent with experimental observations in wild type and mutant yeast cells and suggest that while the ring-septum closure rate is independently set by the septation process, the ring tension serves a vital role by regulating septum growth and thereby suppressing roughness of the septum boundary and maintaining its circular shape. From the model we calculated the minimum ring tension required for a given degree of smoothness in the growing septum, consistent with model computations of wild type fission yeast ring tension. Thus, our model suggests that the primary role of the contractile ring in fission yeast cytokinesis is as a tension-producing machine that regulates septum circularity to ensure ordered closure and separation of the daughter cells.