Understanding the septation initiation network (SIN) function during meiotic cytokinesis in fission yeast

Abstract

Cytokinesis in all organisms involves the creation of membranous barriers that demarcate individual daughter cells. The fission yeast Schizosaccharomyces pombe uses two distinct mechanisms of cytokinesis depending on the mode of cell cycle regulation. During mitosis, it divides using actomyosin ring constriction of which is coordinated with the formation of new membrane/cell wall during cytokinesis. During meiosis, however, four daughter cells (spores) are generated through a unique form of cytokinesis, called sporulation. The de novo synthesis of a double-layered membrane, termed the forespore membrane (FSM) is initiated during meiosis II, which encapsulates the meiotic nuclei. A signalling module termed the Septation Initiation Network (SIN) plays an essential role in the assembly of new membranes and cell wall during mitotic cytokinesis. Krapp et al (2006) has recently shown that SIN proteins localize to SPBs during meiosis and the pathway is activated during meiosis II. Some SIN temperature sensitive mutant cells are defective in FSM assembly which results in unencapsulated nuclei (Krapp et al., 2006). However, as in the case of mitotic cells, the precise mechanism linking SIN and FSM assembly during meiotic cytokinesis is not fully understood. How F-actin participates in this process and whether SIN regulates actin cytoskeletal function during meiosis are yet poorly understood.

In order to study how SIN regulates the FSM assembly, in Chapter III, I investigated the role of a conserved serine-threonine protein kinase Slk1p. Slk1p is analogous to the SIN component Sid2p. Slk1p is expressed specifically during meiosis and localizes to the spindle pole bodies (SPBs) during meiosis I and II in a SIN dependent manner. Slk1p also localizes to the FSM during sporulation. Cells lacking Slk1p display defects associated with sporulation, leading to the formation of asci with smaller and / or fewer spores. The ability of slk1-null cells to sporulate is fully abolished when function of Sid2p is compromised, suggesting that Slk1p and Sid2p play overlapping roles in sporulation. Moreover, increased expression of the syntaxin Psy1p rescues the sporulation defect of sid2-250 slk1-null. Thus, It is likely that Slk1p and Sid2p play a role in FSM assembly by facilitating recruitment of components of the secretory apparatus, such as Psy1p, to allow membrane expansion. These studies thereby suggest that SlN is required to couple the growth of FSM to the meiotic nuclear division.

In order to investigate the role of F-actin and how SIN regulates actin cytoskeleton during meiosis, in Chapter IV, I analyzed the dynamics of F-actin and characterized the role of actin nucleators during sporulation in sin mutant cells. F-actin assembles into 4 ring structures per ascus, referred to as the MeiAR (meiotic actin ring). The actin nucleators Arp2p/3p and formin-For3p assemble into ring structures that overlap with the leading edge protein Meu14p, whereas F-actin makes rings that occupy a larger region behind Meu14p. Time-lapse microscopy shows that the MeiAR assembles near the SPBs and undergoes an expansion in diameter during the early stages of meiosis II, followed by closure in later stages of meiosis II. MeiAR closure completes the process of FSM assembly. Loss of MeiAR leads to excessive assembly of FSMs with a deformed appearance. The rate of closure of the MeiAR is dictated by the function of SIN. These experiments establish the fact that the MeiAR ensures proper targeting of the membrane biogenesis machinery to the leading edge, thereby ensuring the formation of spherically shaped spores.

In summary, this study provides a novel link between the SIN and vesicle trafficking during meiotic cytokinesis. It may also contribute to a better understanding of the coordination among SIN, membrane trafficking and MeiAR in fission yeast as well.