•Spindle pole bodies (SPBs) serve as fungal centrosomes in microtubule nucleation•SPBs are disc-shaped structures associated with the nuclear envelope•Analysis of SPB composition and regulation has ...provided key insights into centrosome function and evolution•Microtubule nucleation is mediated by the γ-tubulin complex, which has been studied biochemically and genetically•Insertion of the SPB into the nuclear envelope requires membrane remodeling
The fungal kingdom is large and diverse, representing extremes of ecology, life cycles and morphology. At a cellular level, the diversity among fungi is particularly apparent at the spindle pole body (SPB). This nuclear envelope embedded structure, which is essential for microtubule nucleation, shows dramatically different morphologies between different fungi. However, despite phenotypic diversity, many SPB components are conserved, suggesting commonalities in structure, function and duplication. Here, I review the organization of the most well-studied SPBs and describe how advances in genomics, genetics and cell biology have accelerated knowledge of SPB architecture in other fungi, providing insights into microtubule nucleation and other processes conserved across eukaryotes.
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.
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.
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.
Nucleation of microtubules by eukaryotic microtubule organizing centers (MTOCs) is required for a variety of functions, including chromosome segregation during mitosis and meiosis, cytokinesis, ...fertilization, cellular morphogenesis, cell motility, and intracellular trafficking. Analysis of MTOCs from different organisms shows that the structure of these organelles is widely varied even though they all share the function of microtubule nucleation. Despite their morphological diversity, many components and regulators of MTOCs, as well as principles in their assembly, seem to be conserved. This review focuses on one of the best-characterized MTOCs, the budding yeast spindle pole body (SPB). We review what is known about its structure, protein composition, duplication, regulation, and functions. In addition, we discuss how studies of the yeast SPB have aided investigation of other MTOCs, most notably the centrosome of animal cells.
DNA double-strand breaks (DSBs) are among the most deleterious forms of DNA lesions in cells. Here we induced site-specific DSBs in yeast cells and monitored chromatin dynamics surrounding the DSB ...using Chromosome Conformation Capture (3C). We find that formation of a DSB within G1 cells is not sufficient to alter chromosome dynamics. However, DSBs formed within an asynchronous cell population result in large decreases in both intra- and interchromosomal interactions. Using live cell microscopy, we find that changes in chromosome dynamics correlate with relocalization of the DSB to the nuclear periphery. Sequestration to the periphery requires the nuclear envelope protein, Mps3p, and Mps3p-dependent tethering delays recombinational repair of a DSB and enhances gross chromosomal rearrangements. Furthermore, we show that components of the telomerase machinery are recruited to a DSB and that telomerase recruitment is required for its peripheral localization. Based on these findings, we propose that sequestration of unrepaired or slowly repaired DSBs to the nuclear periphery reflects a competition between alternative repair pathways.
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.
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