Three-dimensional arrangement of F-actin in the contractile ring of fission yeast

The contractile ring, which is required for cytokinesis in animal and yeast cells, consists mainly of actin filaments. Here, we investigate the directionality of the filaments in fission yeast using myosin S1 decoration and electron microscopy. The contractile ring is composed of around 1,000 to 2,000 filaments each around 0.6 μm in length. During the early stages of cytokinesis, the ring consists of two semicircular populations of parallel filaments of opposite directionality. At later stages, before contraction, the ring filaments show mixed directionality. We consider that the ring is initially assembled from a single site in the division plane and that filaments subsequently rearrange before contraction initiates

Augmin-dependent microtubule nucleation at microtubule walls in the spindle

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Cell Biology 202 (2013): 25-33, doi:10.1083/jcb.201304031.The formation of a functional spindle requires microtubule (MT) nucleation from within the spindle, which depends on augmin. How augmin contributes to MT formation and organization is not known because augmin-dependent MTs have never been specifically visualized. In this paper, we identify augmin-dependent MTs and their connections to other MTs by electron tomography and 3D modeling. In metaphase spindles of human cells, the minus ends of MTs were located both around the centriole and in the body of the spindle. When augmin was knocked down, the latter population of MTs was significantly reduced. In control cells, we identified connections between the wall of one MT and the minus end of a neighboring MT. Interestingly, the connected MTs were nearly parallel, unlike other examples of end–wall connections between cytoskeletal polymers. Our observations support the concept of augmin-dependent MT nucleation at the walls of existing spindle MTs. Furthermore, they suggest a mechanism for maintaining polarized MT organization, even when noncentrosomal MT initiation is widespread.This work was supported by the Next Generation grant (Japan Society for the Promotion of Science), Human Frontier Science Program, James A. and Faith Miller Memorial Fund (to G. Goshima), the Hori Sciences and Arts Foundation, the Sasakawa Scientific Research Grant, the Kazato Research foundation (to T. Kamasaki), and the National Institutes of Health (8P41GM103431-42 to A. Hoenger). T. Kamasaki was a recipient of the Japan Society for the Promotion of Science postdoctoral fellowship.2014-01-0

Bgs1p is responsible for primary septum formation

[EN]Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires coordinated contraction of an actomyosin ring and synthesis of both plasmatic membrane and a septum structure that will constitute the new cell wall end. Schizosaccharomyces pombe contains four essential putative (1,3)β-D-glucan synthase catalytic subunits, Bgs1p to Bgs4p. Here we examined the function of Bgs1p in septation by studying the lethal phenotypes of bgs1 + shut-off and bgs1 Δ cells and demonstrated that Bgs1p is responsible and essential for linear (1,3)β-D-glucan and primary septum formation. bgs1 + shut-off generates a more than 300-fold Bgs1p reduction, but the septa still present large amounts of disorganized linear (1,3)β-D-glucan and partial primary septa. Conversely, both structures are absent in bgs1 Δ cells, where there is no Bgs1p. The septum analysis of bgs1+-repressed cells indicates that linear (1,3)β-D-glucan is necessary but not sufficient for primary septum formation. Linear (1,3)β-D-glucan is the polysaccharide that specifically interacts with the fluorochrome Calcofluor white in fission yeast. We also show that in the absence of Bgs1p abnormal septa are formed, but the cells cannot separate and eventually die

A new membrane protein Sbg1 links the contractile ring apparatus and septum synthesis machinery in fission yeast

Cytokinesis in many organisms requires a plasma membrane anchored actomyosin ring, whose contraction facilitates cell division. In yeast and fungi, actomyosin ring constriction is also coordinated with division septum assembly. How the actomyosin ring interacts with the plasma membrane and the plasma membrane-localized septum synthesizing machinery remains poorly understood. In Schizosaccharomyces pombe, an attractive model organism to study cytokinesis, the β-1,3-glucan synthase Cps1p / Bgs1p, an integral membrane protein, localizes to the plasma membrane overlying the actomyosin ring and is required for primary septum synthesis. Through a high-dosage suppressor screen we identified an essential gene, sbg1+ (suppressor of beta glucan synthase 1), which suppressed the colony formation defect of Bgs1-defective cps1-191 mutant at higher temperatures. Sbg1p, an integral membrane protein, localizes to the cell ends and to the division site. Sbg1p and Bgs1p physically interact and are dependent on each other to localize to the division site. Loss of Sbg1p results in an unstable actomyosin ring that unravels and slides, leading to an inability to deposit a single contiguous division septum and an important reduction of the β-1,3-glucan proportion in the cell wall, coincident with that observed in the cps1-191 mutant. Sbg1p shows genetic and / or physical interaction with Rga7p, Imp2p, Cdc15p, and Pxl1p, proteins known to be required for actomyosin ring integrity and efficient septum synthesis. This study establishes Sbg1p as a key member of a group of proteins that link the plasma membrane, the actomyosin ring, and the division septum assembly machinery in fission yeast

Estudio del mecanismo de supervivencia en ausencia de la actividad esencial ß-(1-3) glucán sintasa

Trabajo presentado en el XV Congreso Nacional de Micología, celebrado en Valencia (España), del 7 al 9 de septiembre de 202

Fission yeast Ags1 confers the essential septum strength needed for safe gradual cell abscission

[EN]Fungal cytokinesis requires the assembly of a dividing septum wall. In yeast, the septum has to be selectively digested during the critical cell separation process. Fission yeast cell wall alpha (1-3) glucan is essential, but nothing is known about its localization and function inthe cell wall or about cooperation between the alpha - and beta (1-3) glucan synthases Ags1 and Bgs for cell wall and septum assembly. Here, we generate a physiological Ags1-GFP variant and demonstrate a tight colocalization with Bgs1, suggesting a cooperation in the important early steps of septum construction. Moreover, we define the essential functions of alpha(1-3) glucan in septation and cell separation. We show that alpha (1-3) glucan is essential for both secondary septum formation and the primary septum structural strength needed to support the physical forces of the cell turgor pressure during cell separation. Consequently, the absence of Ags1 and therefore alpha(1-3)glucan generates a special and unique side-explosive cell separation due to an instantaneous primary septum tearing caused by the turgor pressure

Fission yeast septation

In animal cells cytokinesis relies on the contraction of an actomyosin ring that pulls the plasma membrane to create a cleavage furrow, whose ingression finally divides the mother cell into two daughter cells. Fungal cells are surrounded by a tough and flexible structure called cell wall, which is considered to be the functional equivalent of the extracellular matrix in animal cells. Therefore, in addition to cleavage furrow ingression, fungal cytokinesis also requires the centripetal formation of a septum wall structure that develops between the dividing cells, whose genesis must be strictly coordinated with both the actomyosin ring closure and plasma membrane ingression. Here we briefly review what is known about the septum structure and composition in the fission yeast Schizosaccharomyces pombe, the recent progress about the relationship between septum biosynthesis and actomyosin ring constriction, and the importance of the septum and ring in the steady progression of the cleavage furrow.This work was supported by the Spanish Ministry of Science and Innovation (BFU2010-15641 and BFU2013-39394-P) to PP. JCR was financed by the Spanish Ministry of Science and Innovation (BIO2012-35372 and BIO2015-69958-P), and the Junta de Castilla y León, Spain (CSI037U14). JCGC was supported by a Juan de la Cierva postdoctoral contract from the Spanish Ministry of Science and Innovation.Peer Reviewe

Intracellular Trafficking Pathway of Yeast Long-chain Base Kinase Lcb4, from Its Synthesis to Its Degradation

Sphingoid long-chain base 1-phosphates act as bioactive lipid molecules in eukaryotic cells. In budding yeast, long-chain base 1-phosphates are synthesized mainly by the long-chain base kinase Lcb4. We recently reported that, soon after yeast cells enter into the stationary phase, Lcb4 is rapidly degraded by being delivered to the vacuole in a palmitoylation- and phosphorylation-dependent manner. In this study, we investigated the complete trafficking pathway of Lcb4, from its synthesis to its degradation. After membrane anchoring by palmitoylation at the Golgi apparatus, Lcb4 is delivered to the plasma membrane (PM) through the late Sec pathway and then to the endoplasmic reticulum (ER). The yeast ER consists of a cortical network juxtaposed to the PM (cortical ER) with tubular connections to the nuclear envelope (nuclear ER). Remarkably, the localization of Lcb4 is restricted to the cortical ER. As the cells reach the stationary phase, G1 cell cycle arrest initiates Lcb4 degradation and its delivery to the vacuole via the Golgi apparatus. The protein transport pathway from the PM to the ER found in this study has not been previously reported. We speculate that this novel pathway is mediated by the PM-ER contact

The cell biology of fission yeast septation

In animal cells, cytokinesis requires the formation of a cleavage furrow that divides the cell into two daughter cells. Furrow formation is achieved by constriction of an actomyosin ring that invaginates the plasma membrane. However, fungal cells contain a rigid extracellular cell wall surrounding the plasma membrane; thus, fungal cytokinesis also requires the formation of a special septum wall structure between the dividing cells. The septum biosynthesis must be strictly coordinated with the deposition of new plasma membrane material and actomyosin ring closure and must occur in such a way that no breach in the cell wall occurs at any time. Because of the high turgor pressure in the fungal cell, even a minor local defect might lead to cell lysis and death. Here we review our knowledge of the septum structure in the fission yeast Schizosaccharomyces pombe and of the recent advances in our understanding of the relationship between septum biosynthesis and actomyosin ring constriction and how the two collaborate to build a cross-walled septum able to support the high turgor pressure of the cell. In addition, we discuss the importance of the septum biosynthesis for the steady ingression of the cleavage furrow.This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2010-15641 and BFU2013-39394-P) to P.P. J.C.R. was financed by the Spanish Ministry of Science and Innovation (grants BIO2012-35372 and BIO2015-69958-P) and by the Junta de Castilla y León, Spain (grant CSI037U14). J.C.G.C. was supported by a Juan de la Cierva postdoctoral contract from the Spanish Ministry of Science and Innovation.Peer Reviewe