Changes in the Nuclear Envelope Environment Affect Spindle Pole Body Duplication in Saccharomyces cerevisiae

Abstract

The Saccharomyces cerevisiae nuclear membrane is part of a complex nuclear envelope environment also containing chromatin, integral and peripheral membrane proteins, and large structures such as nuclear pore complexes (NPCs) and the spindle pole body. To study how properties of the nuclear membrane affect nuclear envelope processes, we altered the nuclear membrane by deleting the SPO7 gene. We found that spo7D cells were sickened by the mutation of genes coding for spindle pole body components and that spo7D was synthetically lethal with mutations in the SUN domain gene MPS3. Mps3p is required for spindle pole body duplication and for a variety of other nuclear envelope processes. In spo7D cells, the spindle pole body defect of mps3 mutants was exacerbated, suggesting that nuclear membrane composition affects spindle pole body function. The synthetic lethality between spo7D and mps3 mutants was suppressed by deletion of specific nucleoporin genes. In fact, these gene deletions bypassed the requirement for Mps3p entirely, suggesting that under certain conditions spindle pole body duplication can occur via an Mps3p-independent pathway. These data point to an antagonistic relationship between nuclear pore complexes and the spindle pole body. We propose a model whereby nuclear pore complexes either compete with the spindle pole body for insertion into the nuclear membrane or affect spindle pole body duplication by altering the nuclear envelope environment. T HE nuclear envelope is composed of distinct outer and inner nuclear membranes. The outer nuclear membrane is continuous with the endoplasmic reticulum. The inner nuclear membrane is associated with a unique set of proteins, some of which mediate interactions between the nuclear envelope and chromatin (reviewed in Zhao et al. 2009). Nuclear pore complexes traverse both membranes and allow transport of proteins and solutes between the cytoplasm and the nucleus. The inner and outer nuclear membranes fuse in the region surrounding each nuclear pore complex.In animal cells, the nuclear envelope disassembles as cells enter mitosis and reassembles upon mitotic exit. Nuclear envelope breakdown allows the association of chromosomes with spindle microtubules, which are nucleated from centrosomes that reside in the cytoplasm. In contrast, certain types of fungi, such as the budding yeast Saccharomyces cerevisiae, undergo closed mitosis, where the nuclear envelope remains intact throughout the entire cell cycle. Closed mitosis is possible because the yeast centrosome-equivalent, the spindle pole body (SPB), is embedded in the nuclear envelope, allowing the SPB to nucleate both cytoplasmic and nuclear microtubules. SPB duplication requires a mechanism for inserting the new SPB into the nuclear envelope (reviewed in Jaspersen and Winey 2004). The new SPB begins to form in late G 1 /early S phase as satellite material deposited on the cytoplasmic face of an electron-dense region of the nuclear envelope, called the half-bridge. The satellite material matures into a dupl...

Mitotic Spindle Form and Function

“…This includes the intriguing finding that the null allele of the essential MPS2 gene can be rescued by deletion of Pom152 or Pom34 (Sezen et al 2009). Similarly, the deletion of Pom152 or the nucleoporin Nup157 will rescue the deletion of the MPS3 gene, another essential SPB membrane protein (Witkin et al 2010). Finally, a screen for SPB-remodeling factors, described below, identified a role for Pom152 in controlling Figure 7 The SPB duplication pathway.…”

“…These interactions between SPBs and NPCs are complex and poorly understood, but involve shared components (Chial et al 1998), shared environment in the nuclear envelope (Witkin et al 2010), and coordinated translational control of component synthesis (Sezen et al 2009).…”

Evidence for actin dual role in regulating chromosome organization and dynamics in yeast

“…This phenotype can be rescued in a mutant of the NPC, Δpom152 ( Fig. S2; Witkin et al, 2010). Yet, Δpom152 itself has an effect on subtelomere organization (Fig.…”

Integrity and Function of theSaccharomyces cerevisiaeSpindle Pole Body Depends on Connections Between the Membrane Proteins Ndc1, Rtn1, and Yop1

“…The SPB is then inserted into a pore in the NE, allowing for assembly of nuclear components to create duplicated side-by-side SPBs (Byers and Goetsch 1974;Byers and Goetsch 1975;Adams and Kilmartin 1999;Jaspersen and Winey 2004;Winey and Bloom 2012). The membrane anchors and half-bridge components both play a role in this SPB insertion step (Winey et al 1991(Winey et al , 1993Schramm et al 2000;Araki et al 2006;Sezen et al 2009;Witkin et al 2010;Friederichs et al 2011;Kupke et al 2011;Winey and Bloom 2012). Unlike NPC assembly, SPB duplication is spatially and temporally restricted.…”

“…One of the SPB membrane anchors is Ndc1, a conserved integral membrane protein that is also an essential NPC Pom and required for NPC assembly Mansfeld et al 2006;Stavru et al 2006;Kind et al 2009). Some NPC components are required for proper remodeling of SPB core components and regulation of SPB size (Niepel et al 2005;Greenland et al 2010), whereas the loss of other NPC components rescues SPB mutant assembly phenotypes Sezen et al 2009;Witkin et al 2010). The exact mechanism by which all of these NPC components influence SPB assembly is not known.…”