Scott C. Schuyler

Microtubule "plus-end-tracking proteins" : The end is just the beginning

There are several key unanswered questions about the +TIPs. One is the mechanism of release of +TIPs from the trailing edge of polymerizing MTs. An important barrier to progress is the absence of an in vitro system that faithfully recapitulates both the binding and release that characterize plus end tracking. As +TIP partners/regulators may be important to reconstitute +TIP treadmilling in vitro, future progress may come from systems with additional purified components, or from ones based on crude extracts. Another question is the in vivo relationship between different +TIPs. It appears that CLIP-170 and EB1 can reside on the same growing MT end (Akhmanova et al., 2001xAkhmanova, A., Hoogenraad, C.C., Drabek, K., Stepanova, T., Dortland, B., Verkerk, T., Vermeulen, W., Burgering, B.M., De Zeeuw, C.I., Grosveld, F., and Galjart, N. Cell. 2001; 104: 923–935Abstract | Full Text | Full Text PDF | PubMed | Scopus (290)See all References)(Akhmanova et al., 2001). Is there cooperation or competition between these proteins, and could such interactions be a nodal point for regulating the repertoire of plus end behaviors?In addition, it is not known whether +TIPs can provide stable attachments. It will be important to determine if their association with MTs is stabilized (i.e., if they treadmill less) when MT plus ends interact with target sites. An alternative, suggested for CLIP-170 at the kinetochore, is that +TIPs mediate the initial attachment but then dissociate (Dujardin et al., 1998xDujardin, D., Wacker, U.I., Moreau, A., Schroer, T.A., Rickard, J.E., and De Mey, J.R. J. Cell Biol. 1998; 141: 849–862Crossref | Scopus (116)See all References)(Dujardin et al., 1998). Real-time methods where the turnover of +TIPs at the MT end can be measured may help distinguish between these possibilities. Although budding yeast is not famous for the awesome power of its cytology, the ability to see single MTs interacting with target sites on the membrane provides unique spatial resolution that should facilitate these experiments. Finally, do the +TIPs only function at the MT plus end? Several +TIPs interact with proteins that associate with or regulate the behavior of the MT minus ends (Chen et al. 1998xChen, X.P., Yin, H., and Huffaker, T.C. J. Cell Biol. 1998; 141: 1169–1179Crossref | Scopus (71)See all References, Chen et al. 2000xChen, C.R., Chen, J., and Chang, E.C. Mol. Biol. Cell. 2000; 11: 4067–4077CrossrefSee all References). This raises the possibility that +TIPs might have an additional role in MT nucleation or in anchoring MT minus ends to the centrosome.Like motors and the proteins involved in microtubule nucleation, the CLIP-170 family and EB1 family proteins are highly conserved. We speculate that these proteins evolved because of the need for devices that distinguish the plus end from the body of the MT. Although the plus end has a unique shape, its large size (>25 nm in diameter) makes it an unwieldy object to recognize at the molecular level. +TIPs may solve this problem; if copolymerized with tubulin, +TIPs may “tag” MT plus ends. By interacting with different partners, +TIPs could serve as molecular adaptors, providing links to a large repertoire of signals and target sites. In the cell, this diversity of plus end interactions may be fundamental to the regional control of MT dynamics, MT attachment, and the assembly of complex MT-based structures necessary for cell division and morphogenesis.

The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix

The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B). Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function. Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs. By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer. Ase1p bound and bundled MTs in vitro. By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase. Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase–arrested cells. FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs. We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone. MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix.

THE APC-ASSOCIATED PROTEIN EB1 ASSOCIATES WITH COMPONENTS OF THE DYNACTIN COMPLEX AND CYTOPLASMIC DYNEIN INTERMEDIATE CHAIN

Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein [1], a domain of APC that is commonly deleted in colorectal neoplasia [2]. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 [3] and Saccharomyces cerevisiae Bim1p [4]. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex [5]. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle [6], in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis [7] [8] [9] [10]. Here, we demonstrate that human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 was also found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sedimented at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex was independent of APC and was preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes.

The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. Microtubule-associated proteins.

The midzone is the domain of the mitotic spindle that maintains spindle bipolarity during anaphase and generates forces required for spindle elongation (anaphase B). Although there is a clear role for microtubule (MT) motor proteins at the spindle midzone, less is known about how microtubule-associated proteins (MAPs) contribute to midzone organization and function. Here, we report that budding yeast Ase1p is a member of a conserved family of midzone-specific MAPs. By size exclusion chromatography and velocity sedimentation, both Ase1p in extracts and purified Ase1p behaved as a homodimer. Ase1p bound and bundled MTs in vitro. By live cell microscopy, loss of Ase1p resulted in a specific defect: premature spindle disassembly in mid-anaphase. Furthermore, when overexpressed, Ase1p was sufficient to trigger spindle elongation in S phase–arrested cells. FRAP revealed that Ase1p has both a very slow rate of turnover within the midzone and limited lateral diffusion along spindle MTs. We propose that Ase1p functions as an MT cross-bridge that imparts matrix-like characteristics to the midzone. MT-dependent networks of spindle midzone MAPs may be one molecular basis for the postulated spindle matrix.

Japanese encephalitis virus co-opts the ER-stress response protein GRP78 for viral infectivity.

The serum-free medium from Japanese encephalitis virus (JEV) infected Baby Hamster Kidney-21 (BHK-21) cell cultures was analyzed by liquid chromatography tandem mass spectrometry (LC-MS) to identify host proteins that were secreted upon viral infection. Five proteins were identified, including the molecular chaperones Hsp90, GRP78, and Hsp70. The functional role of GRP78 in the JEV life cycle was then investigated. Co-migration of GRP78 with JEV particles in sucrose density gradients was observed and co-localization of viral E protein with GRP78 was detected by immunofluorescence analysis in vivo. Knockdown of GRP78 expression by siRNA did not effect viral RNA replication, but did impair mature viral production. Mature viruses that do not co-fractionate with GPR78 displayed a significant decrease in viral infectivity. Our results support the hypothesis that JEV co-opts host cell GPR78 for use in viral maturation and in subsequent cellular infections.The serum-free medium from Japanese encephalitis virus (JEV) infected Baby Hamster Kidney-21 (BHK-21) cell cultures was analyzed by liquid chromatography tandem mass spectrometry (LC-MS) to identify host proteins that were secreted upon viral infection. Five proteins were identified, including the molecular chaperones Hsp90, GRP78, and Hsp70. The functional role of GRP78 in the JEV life cycle was then investigated. Co-migration of GRP78 with JEV particles in sucrose density gradients was observed and co-localization of viral E protein with GRP78 was detected by immunofluorescence analysis in vivo. Knockdown of GRP78 expression by siRNA did not effect viral RNA replication, but did impair mature viral production. Mature viruses that do not co-fractionate with GPR78 displayed a significant decrease in viral infectivity. Our results support the hypothesis that JEV co-opts host cell GPR78 for use in viral maturation and in subsequent cellular infections.

The Mad1–Mad2 balancing act – a damaged spindle checkpoint in chromosome instability and cancer

Summary Cancer cells are commonly aneuploid. The spindle checkpoint ensures accurate chromosome segregation by controlling cell cycle progression in response to aberrant microtubule–kinetochore attachment. Damage to the checkpoint, which is a partial loss or gain of checkpoint function, leads to aneuploidy during tumorigenesis. One form of damage is a change in levels of the checkpoint proteins mitotic arrest deficient 1 and 2 (Mad1 and Mad2), or in the Mad1:Mad2 ratio. Changes in Mad1 and Mad2 levels occur in human cancers, where their expression is regulated by the tumor suppressors p53 and retinoblastoma 1 (RB1). By employing a standard assay, namely the addition of a mitotic poison at mitotic entry, it has been shown that checkpoint function is normal in many cancer cells. However, in several experimental systems, it has been observed that this standard assay does not always reveal checkpoint aberrations induced by changes in Mad1 or Mad2, where excess Mad1 relative to Mad2 can lead to premature anaphase entry, and excess Mad2 can lead to a delay in entering anaphase. This Commentary highlights how changes in the levels of Mad1 and Mad2 result in a damaged spindle checkpoint, and explores how these changes cause chromosome instability that can lead to aneuploidy during tumorigenesis.

Analysis of the size and shape of protein complexes from yeast

Publisher Summary This chapter presents the analysis of the size and shape of protein complexes from yeast and describes the way for estimating the molecular weight and shape of soluble proteins from yeast. These estimates—when coupled with epitope tagging and coimmunoprecipitation—give insight into the physical properties and molecular makeup of protein complexes. These estimates can also be very informative when pure recombinant protein is compared with the native protein. The strength of these methods rests in the fact that knowledge of only a single biochemical activity—even as simple as a western blot—is sufficient to gain some insight into molecular composition and structure. These methods may also be applied to biological complexes with mixed classes of components such as protein–RNA complexes. The results of size and shape measurement are quantitative, and thus can be compared directly with previously determined experimental values. Such comparisons can be very powerful and can provide insight into molecular structure and function.

The Hsp90-dependent proteome is conserved and enriched for hub proteins with high levels of protein-protein connectivity

Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic and nongenetic variation within a population. Despite information about Hsp90 protein–protein interactions, a global view of the Hsp90-regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the “stable isotope labeling by amino acids in cell culture” method coupled with mass spectrometry to quantify around 4,000 proteins in low-Hsp90 cells. We observed that 904 proteins changed in their abundance by more than 1.5-fold. When compared with the transcriptome of the same population of cells, two-thirds of the misregulated proteins were observed to be affected posttranscriptionally, of which the majority were downregulated. Further analyses indicated that the downregulated proteins are highly conserved and assume central roles in cellular networks with a high number of protein interacting partners, suggesting that Hsp90 buffers genetic and nongenetic variation through regulating protein network hubs. The downregulated proteins were enriched for essential proteins previously not known to be Hsp90-dependent. Finally, we observed that downregulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response, respectively, providing a direct link between observed proteome regulation and cellular phenotypes.

Reduced Mad2 expression keeps relaxed kinetochores from arresting budding yeast in mitosis

A study of the consequences of removing one copy of Mad2 in diploid budding yeast shows that MAD2 haploinsufficiency is due to subunit imbalance in the Mad1–Mad2 complex and that a normal Mad2:Mad1 ratio is essential for cells to respond to a loss of tension on mitotic chromosomes but is dispensable for a response to unattached chromosomes.

Functional Impact of RNA editing and ADARs on regulation of gene expression: perspectives from deep sequencing studies

Cells regulate gene expression at multiple levels leading to a balance between robustness and complexity within their proteome. One core molecular step contributing to this important balance during metazoan gene expression is RNA editing, such as the co-transcriptional recoding of RNA transcripts catalyzed by the adenosine deaminse acting on RNA (ADAR) family of enzymes. Understanding of the adenosine-to-inosine RNA editing process has been broadened considerably by the next generation sequencing (NGS) technology, which allows for in-depth demarcation of an RNA editome at nucleotide resolution. However, critical issues remain unresolved with regard to how RNA editing cooperates with other transcript-associated events to underpin regulated gene expression. Here we review the growing body of evidence, provided by recent NGS-based studies, that links RNA editing to other mechanisms of post-transcriptional RNA processing and gene expression regulation including alternative splicing, transcript stability and localization, and the biogenesis and function of microRNAs (miRNAs). We also discuss the possibility that systematic integration of NGS data may be employed to establish the rules of an “RNA editing code”, which may give us new insights into the functional consequences of RNA editing.