Proteome Analysis of the Human Mitotic Spindle

Sauer, Guido; Körner, Roman; Hanisch, Anja; Ries, Albert; Nigg, Erich A.; Silljé, Herman H W

doi:10.1074/mcp.m400158-mcp200

Cited by 232 publications

(269 citation statements)

References 48 publications

Supporting

Mentioning

249

Contrasting

Order By: Relevance

“…1) (34). Taxolstabilized mitotic spindles (including kinetochores and centrosomes) were purified as described (34), and spindle proteins were separated on one-dimensional SDS͞PAGE gradient gels. After Coomassie blue staining, each lane was cut into 18 pieces, covering different protein mass ranges.…”

Section: Resultsmentioning

confidence: 99%

“…Taxolstabilized mitotic spindles (including kinetochores and centrosomes) were purified as described in ref. 34, except that okadaic acid (100 nM) was added to all buffers to prevent dephosphorylation by PP2A-and PP1-type protein phosphatases. In short, HeLa S3 cells were synchronized in mitosis by using a sequential aphidicolin block͞release, nocodazole block protocol.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we have purified mitotic spindles from human HeLa S3 cells and identified a total of 795 proteins, including 151 previously known spindle-associated components (34). In this former study, we have also characterized the purity of the spindle preparations, using differential interference contrast microscopy, Western blot analyses with antibodies against known spindle and control proteins, tandem mass spectrometry (MS͞ MS), and cell biological validation experiments.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Phosphoproteome analysis of the human mitotic spindle

Nousiainen

Silljé

Sauer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

303

260

View full text Add to dashboard Cite

During cell division, the mitotic spindle segregates the sister chromatids into two nascent cells, such that each daughter cell inherits one complete set of chromosomes. Errors in spindle formation can result in both chromosome missegregation and cytokinesis defects and hence lead to genomic instability. To ensure the correct function of the spindle, the activity and localization of spindle associated proteins has to be tightly regulated in time and space. Reversible phosphorylation has been shown to be one of the key regulatory mechanisms for the organization of the mitotic spindle. The relatively low number of identified in vivo phosphorylation sites of spindle components, however, has hampered functional analysis of regulatory spindle networks. A more complete inventory of the phosphorylation sites of spindle-associated proteins would therefore constitute an important advance. Here, we describe the mass spectrometry-based identification of in vivo phosphorylation sites from purified human mitotic spindles. In total, 736 phosphorylation sites were identified, of which 312 could be attributed to known spindle proteins. Among these are phosphorylation sites that were previously shown to be important for the regulation of spindle-associated proteins. Importantly, this data set also comprises 279 novel phosphorylation sites of known spindle proteins for future functional studies. This inventory of spindle phosphorylation sites should thus make an important contribution to a better understanding of the molecular mechanisms that regulate the formation, function, and integrity of the mitotic spindle. mass spectrometry ͉ mitosis ͉ molecular cell biology ͉ phosphorylation ͉ proteomics A t the transition from interphase to mitosis, the microtubule network undergoes a profound change that culminates in the formation of the spindle apparatus. The mitotic spindle then serves to segregate the chromosomes to opposite poles of the cell and to define the plane of cell division. A large number of proteins associate with this microtubule-based structure and regulate its dynamic formation and function (1, 2). Several spindle proteins, including XKCM1 and XMAP215 family members (3, 4), have been identified that either stabilize or destabilize microtubules by mediating the rapid changes between polymerization and depolymerization (5, 6). These proteins play an important role in spindle formation, as illustrated by the striking change in microtubule half-life from 5-10 min in interphase to less than 1 min in mitosis (7), which results in the short and unstable microtubules characteristic of mitosis (5, 6). Another important group of spindle proteins comprises motors of the kinesin and dynein families that are essential for mitotic progression (5,6,8,9). They push the spindle poles away from each other during early mitosis and play crucial roles in capturing chromosomes and positioning them at the metaphase plate. Subsequently, motors also contribute to central spindle formation and cytokinesis. In addition, numerous structural prote...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Phosphoproteome analysis of the human mitotic spindle

Nousiainen

Silljé

Sauer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

303

260

View full text Add to dashboard Cite

show abstract

“…From the primary function of DNA photolyases, namely repairing DNA lesions, interaction with DNA is crucial, but interactions with spindle structures consisting of microtubules cannot currently be explained. However, it is known that many proteins bind to the spindles; among these are also nucleic acid-binding proteins (Sauer et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

DNA photolyases of Chrysodeixis chalcites nucleopolyhedrovirus are targeted to the nucleus and interact with chromosomes and mitotic spindle structures

Fang

Vlak

Eker

et al. 2009

Journal of General Virology

View full text Add to dashboard Cite

Cyclobutane pyrimidine dimer (CPD) photolyases convert UV-induced CPDs in DNA into monomers using visible light as the energy source. Two phr genes encoding class II CPD photolyases PHR1 and PHR2 have been identified in Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV). Transient expression assays in insect cells showed that PHR1-EGFP fusion protein was localized in the nucleus. Early after transfection, PHR2-EGFP was distributed over the cytoplasm and nucleus but, over time, it became localized predominantly in the nucleus. Immunofluorescence analysis with anti-PHR2 antiserum showed that, early after transfection, non-fused PHR2 was already present mainly in the nucleus, suggesting that the fusion of PHR2 to EGFP hindered its nuclear import. Both PHR-EGFP fusion proteins strongly colocalized with chromosomes and spindle, aster and midbody structures during host-cell mitosis. When PHR2-EGFP-transfected cells were superinfected with Autographa californica multiplenucleocapsid NPV (AcMNPV), the protein colocalized with virogenic stroma, the replication factories of baculovirus DNA. The collective data support the supposition that the PHR2 protein plays a role in baculovirus DNA repair.

show abstract

“…By proteomic analysis, nucleolin has been identified in human centrosomes 15 and mitotic spindles. 30 By immunofluorescence, a phosphorylated form of nucleolin has also been detected in mitosis at the spindle poles 28 but its function in these structures has never been addressed.…”

Section: Introductionmentioning

confidence: 99%

Centrosomal nucleolin is required for microtubule network organization

et al. 2015

View full text Add to dashboard Cite

Nucleolin is a pleiotropic protein involved in a variety of cellular processes. Although multipolar spindle formation has been observed after nucleolin depletion, the roles of nucleolin in centrosome regulation and functions have not been addressed. Here we report using immunofluorescence and biochemically purified centrosomes that nucleolin colocalized only with one of the centrioles during interphase which was further identified as the mature centriole. Upon nucleolin depletion, cells exhibited an amplification of immature centriole markers surrounded by irregular pericentrin staining; these structures were exempt from maturation markers and unable to nucleate microtubules. Furthermore, the microtubule network was disorganized in these cells, exhibiting frequent non-centrosomal microtubules. At the mature centriole a reduced kinetics in the centrosomal microtubule nucleation phase was observed in live silenced cells, as well as a perturbation of microtubule anchoring. Immunoprecipitation experiments showed that nucleolin belongs to protein complexes containing 2 key centrosomal proteins, g-tubulin and ninein, involved in microtubule nucleation and anchoring steps. Altogether, our study uncovered a new role for nucleolin in restricting microtubule nucleation and anchoring at centrosomes in interphase cells.

show abstract

Proteome Analysis of the Human Mitotic Spindle

Cited by 232 publications

References 48 publications

Phosphoproteome analysis of the human mitotic spindle

Phosphoproteome analysis of the human mitotic spindle

DNA photolyases of Chrysodeixis chalcites nucleopolyhedrovirus are targeted to the nucleus and interact with chromosomes and mitotic spindle structures

Centrosomal nucleolin is required for microtubule network organization

Contact Info

Product

Resources

About