Centrosomes are a functionally conserved feature of eukaryotic cells that play an important role in cell division. The conserved γ-tubulin complex organizes spindle and astral microtubules, which, in ...turn, separate replicated chromosomes accurately into daughter cells. Like DNA, centrosomes are duplicated once each cell cycle. Although in some cell types it is possible for cell division to occur in the absence of centrosomes, these divisions typically result in defects in chromosome number and stability. In single-celled organisms such as fungi, centrosomes known as spindle pole bodies (SPBs) are essential for cell division. SPBs also must be inserted into the membrane because fungi undergo a closed mitosis in which the nuclear envelope (NE) remains intact. This poorly understood process involves events similar or identical to those needed for de novo nuclear pore complex assembly. Here, we review how analysis of fungal SPBs has advanced our understanding of centrosomes and NE events.
Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely ...unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.
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•Mitotic transcription inhibition occurs in early mitosis•P-TEFb is required for mitotic transcriptional activation and release of paused Pol II•Nascent RNA-seq and RNA FISH reveal active transcription at the onset of mitosis•Inhibition of mitotic transcriptional activation delays cell-cycle progression
How transcription is shut down as cells begin to condense chromosomes during mitosis is poorly understood. Liang et al. report the requirement of mitotic transcriptional activation by P-TEFb to release paused Pol II as a prerequisite for this process, and ultimately for proper cell-cycle progression.
Protein quality control and transport are important for the integrity of organelles such as the endoplasmic reticulum, but it is largely unknown how protein homeostasis is regulated at the nuclear ...envelope (NE) despite the connection between NE protein function and human disease. Elucidating mechanisms that regulate the NE proteome is key to understanding nuclear processes such as gene expression, DNA replication and repair as NE components, particularly proteins at the inner nuclear membrane (INM), are involved in the maintenance of nuclear structure, nuclear positioning and chromosome organization. Nuclear pore complexes control the entry and exit of proteins in and out of the nucleus, restricting movement across the nuclear membrane based on protein size, or the size of the extraluminal-facing domain of a transmembrane protein, providing one level of INM proteome regulation. Research in budding yeast has identified a protein quality control system that targets mislocalized and misfolded proteins at the INM. Here, we review what is known about INM-associated degradation, including recent evidence suggesting that it not only targets mislocalized or misfolded proteins, but also contributes to homeostasis of resident INM proteins.
The expression of genes residing near telomeres is attenuated through telomere position-effect variegation (TPEV). By using a
URA3 reporter located at
TEL-VII-L of
Saccharomyces cerevisiae, it was ...proposed that the disruptor of telomeric silencing-1 (Dot1) regulates TPEV by catalyzing H3K79 methylation.
URA3 reporter assays also indicated that H3K79 methylation is required for
HM silencing. Surprisingly, a genome-wide expression analysis of H3K79 methylation-defective mutants identified only a few telomeric genes, such as
COS12 at
TEL-VII-L, to be subject to H3K79 methylation-dependent natural silencing. Consistently, loss of Dot1 did not globally alter Sir2 or Sir3 occupancy in subtelomeric regions, but only led to some telomere-specific changes. Furthermore, H3K79 methylation by Dot1 did not play a role in the maintenance of natural
HML silencing. Therefore, commonly used
URA3 reporter assays may not report on natural PEV, and therefore, studies concerning the epigenetic mechanism of silencing in yeast should also employ assays reporting on natural gene expression patterns.
► Maintenance of natural telomeric silencing does not require Dot1 or H3K79 methylation ► Loss of Dot1 did not globally alter Sir2 or Sir3 occupancy in subtelomeric regions ► Dot1 is not required for the maintenance of natural HML silencing ►
URA3 reporter located at
TEL-VII-L or HM loci may not report on natural PEV
Highlights • The nuclear envelope (NE) is actively remodeled during mitosis. • Microtubule-organizing centers (MTOCs) insert into the NE in closed mitosis. • The NE disassembles during open mitosis ...to facilitate spindle formation. • Membrane bending, composition and stabilization are associated with MTOCs.
Microtubule-organizing centers (MTOCs), known as centrosomes in animals and spindle pole bodies (SPBs) in fungi, are important for the faithful distribution of chromosomes between daughter cells ...during mitosis as well as for other cellular functions. The cytoplasmic duplication cycle and regulation of the
SPB is analogous to centrosomes, making it an ideal model to study MTOC assembly. Here, we use superresolution structured illumination microscopy with single-particle averaging to localize 14
SPB components and regulators, determining both the relationship of proteins to each other within the SPB and how each protein is assembled into a new structure during SPB duplication. These data enabled us to build the first comprehensive molecular model of the
SPB, resulting in structural and functional insights not ascertained through investigations of individual subunits, including functional similarities between Ppc89 and the budding yeast SPB scaffold Spc42, distribution of Sad1 to a ring-like structure and multiple modes of Mto1 recruitment.
Saccharomyces cerevisiae is an ideal model eukaryotic system for the systematic analysis of gene function due to the ease and precision with which its genome can be manipulated. The ability of ...budding yeast to undergo efficient homologous recombination with short stretches of sequence homology has led to an explosion of PCR-based methods to delete and mutate yeast genes and to create fusions to epitope tags and fluorescent proteins. Here, we describe commonly used methods to generate gene deletions, to integrate mutated versions of a gene into the yeast genome, and to construct N- and C-terminal gene fusions. Using a high-efficiency yeast transformation protocol, DNA fragments with as little as 40 bp of homology can accurately target integration into a particular region of the yeast genome.
Proper mitotic progression in
requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB-yeast centrosome) to build the mitotic spindle. Linkage of the centromere ...to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high-resolution structured illumination microscopy, we show that the conserved Sad1-UNC-84 homology-domain protein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for localized NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for redistribution of other SPB ring proteins and for complete NEBD at the SPB to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate partial NEBD and spindle formation through building of a SPB ring structure.
The nuclear envelope (NE) undergoes dynamic remodeling to maintain NE integrity, a process involving the inner nuclear membrane protein LEM2 recruiting CHMP7/Cmp7 and then ESCRT-III. However, prior ...work has hinted at CHMP7/ESCRT-independent mechanisms. To identify such mechanisms, we studied NE assembly in Schizosaccharomyces japonicus, a fission yeast that undergoes partial mitotic NE breakdown and reassembly. S. japonicus cells lacking Cmp7 have compromised NE sealing after mitosis but are viable. A genetic screen identified mutations that promote NE integrity in cmp7Δ cells. Unexpectedly, loss of Lem2 or its interacting partner Nur1 suppressed cmp7Δ defects. In the absence of Cmp7, Lem2 formed aggregates that appear to interfere with ESCRT-independent NE sealing. A gain-of-function mutation implicated a membrane and ESCRT-III regulator, Alx1, in this alternate pathway. Additional results suggest a potentially general role for unsaturated fatty acids in NE integrity. These findings establish the existence of mechanisms for NE sealing independent of the canonical ESCRT pathway.
Bipolar spindle formation in yeast requires insertion of centrosomes (known as spindle pole bodies SPBs) into fenestrated regions of the nuclear envelope (NE). Using structured illumination ...microscopy and bimolecular fluorescence complementation, we map protein distribution at SPB fenestrae and interrogate protein-protein interactions with high spatial resolution. We find that the Sad1-UNC-84 (SUN) protein Mps3 forms a ring-like structure around the SPB, similar to toroids seen for components of the SPB insertion network (SPIN). Mps3 and the SPIN component Mps2 (a Klarsicht-ANC-1-Syne-1 domain KASH-like protein) form a novel noncanonical linker of nucleoskeleton and cytoskeleton (LINC) complex that is connected in both luminal and extraluminal domains at the site of SPB insertion. The LINC complex also controls the distribution of a soluble SPIN component Bbp1. Taken together, our work shows that Mps3 is a fifth SPIN component and suggests both direct and indirect roles for the LINC complex in NE remodeling.
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