Endocytosis in fission yeast is spatially associated with the actin cytoskeleton during polarised cell growth and cytokinesis

Abstract

In the fission yeast, Schizosaccharomyces pombe, uptake of the fluorescent styryl dye FM4-64 via the endocytic pathway to the vacuole was localised to the poles of growing, interphase cells and to the cell equator during cell division, regions of cell wall deposition that are rich in actin. When the pattern of growth or the plane of cytokinesis was altered, the relationship between the actin cytoskeleton and the site of endocytosis was maintained. Transfer of the label to the vacuolar membrane was dependent upon the Rab GTPase Ypt7 and, hence, vesicle fusion. Endocytic vesicles transiently colocalised with actin patches and endocytosis was inhibited in mutants that affected actin patch integrity and by the actin inhibitor latrunculin A. Concentrations of latrunculin that removed actin cables but left patches unaffected had no effect on endocytosis at the poles, but abolished endocytosis at the cell equator. Equatorial, but not polar, endocytosis was also inhibited in cells lacking the formin For3 (which have selectively destabilised actin cables), in mutants of the exocyst complex and in cells treated with brefeldin A. Differential effects on endocytosis at the cell poles and equator were also observed in the actin mutant cps8 and the Arp2/3 complex mutant arp2. The redirection of endocytosis from the cell poles to the cell equator in M phase coincided with the anaphase separation of sister chromatids and was abolished in the septation initiation network (SIN) mutants cdc7, sid1 and sid2, demonstrating that the spatial reorganisation of the endocytic pathway in the S. pombe cell cycle requires a functional SIN pathway. We conclude that endocytosis in fission yeast has two distinct components, both of which are actin-based, but which are mechanistically distinct, as well as being spatially and temporally separated in the S. pombe cell cycle.

A novel interplay between GEFs orchestrates Cdc42 activity during cell polarity and cytokinesis

“…Gef1-mNG persists on both sides of the septum barrier in 40% of cells treated with LatA, but not in mock DMSO-treated cells ( Figure 3C). Cells undergoing ring constriction and septum formation display actin cables as well as Arp2/3-complex-dependent patches at the division site (Coffman et al, 2013;Gachet and Hyams, 2005;Huang et al, 2012;Wang et al, 2016). LatA treatment removes all types of filamentous actin structures (Spector et al, 1983).…”

Atg20- and Atg24-family proteins promote organelle autophagy in fission yeast

“…These sites are presumably plasma membrane regions where polarized exocytosis occurs. In addition, we also observed cytoplasmic puncta of Syb1, some of which could represent endosomes through which Syb1 traffics during its recycling (Gachet and Hyams, 2005;He et al, 2006;Kita et al, 2004). Wild-type-like Syb1 localization patterns were observed in single-mutant cells that lacked Atg20, Atg24b or Mug186.…”

Expression of budding yeast IPT1 produces mannosyldiinositol phosphorylceramide in fission yeast and inhibits cell growth

“…Actin patches assemble at sites of endocytosis, accompanying polarized cell growth at the tips of interphase cells and at the centre of dividing cells (Gachet & Hyams, 2005;Arellano et al, 1997;Balasubramanian et al, 1998;Kovar et al, 2011). Their distribution changes throughout the cell cycle.…”