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Papers by Dannel McCollum

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Nov 10, 2008

Cytokinesis is powered by the contraction of actomyosin filaments within the newly assembled cont... more Cytokinesis is powered by the contraction of actomyosin filaments within the newly assembled contractile ring. Microtubules are a spindle component that is essential for the induction of cytokinesis. This induction could use central spindle and/or astral microtubules to stimulate cortical contraction around the spindle equator (equatorial stimulation). Alternatively, or in addition, induction could rely on astral microtubules to relax the polar cortex (polar relaxation). To investigate the relationship between microtubules, cortical stiffness, and contractile ring assembly, we used different configurations of microtubules to manipulate the distribution of actin in living silkworm spermatocytes. Mechanically repositioned, noninterdigitating microtubules can induce redistribution of actin at any region of the cortex by locally excluding cortical actin filaments. This cortical flow of actin promotes regional relaxation while increasing tension elsewhere (normally at the equatorial cortex). In contrast, repositioned interdigitating microtubule bundles use a novel mechanism to induce local stimulation of contractility anywhere within the cortex; at the antiparallel plus ends of central spindle microtubules, actin aggregates are rapidly assembled de novo and transported laterally to the equatorial cortex. Relaxation depends on microtubule dynamics but not on RhoA activity, whereas stimulation depends on RhoA activity but is largely independent of microtubule dynamics. We conclude that polar relaxation and equatorial stimulation mechanisms redundantly supply actin for contractile ring assembly, thus increasing the fidelity of cleavage.

Springer eBooks, 2013

S-adenosylmethionine (Fig. 1) is synthesized from ATP and L-methionine and acts as a donor of met... more S-adenosylmethionine (Fig. 1) is synthesized from ATP and L-methionine and acts as a donor of methyl groups in prokaryote as well as eukaryote. By the transfer of a methyl group, S-adenosylmethionine is converted into S-adenosylhomocysteine, which is broken down to homocysteine and adenosine. Homocysteine is then converted to methionine. S-adenosylmethionine is also used for synthesis of cysteine as well as polyamines.

Current Biology, Jun 1, 2010

simply, we might simply double the term for the relative siring success of males compared with he... more simply, we might simply double the term for the relative siring success of males compared with hermaphrodites (Figure 2). The previous implausibility of androdioecy in P. angustifolia was thus not due to the unavailability of an appropriate theoretical framework, but rather to a violation of the simple implicit assumption that all hermaphrodites are potential sires for all progeny. The genetic details exposed by Saumitou-Laprade et al.'s study [10] are fascinating and unexpected, but androdioecy in P. angustifolia can be explained by the old paradigm after all, which simply compares the expected relative siring prospects of different sexual phenotypes.

Current Biology, Aug 1, 2005

Medical Center lus in interphase and the spindle during anaphase (Figure 1C) as well as the kinet... more Medical Center lus in interphase and the spindle during anaphase (Figure 1C) as well as the kinetochores in metaphase Worcester, Massachusetts 01605 (Figure 1F; see Figure S1 in the Supplemental Data available with this article online). Both Clp1p-GFP-NES and Summary Clp1p-GFP-NLS presumably still shuttle across the nuclear envelope, but their steady-state levels are shifted Cdc14-like phosphatases regulate a variety of cell cyto either the cytoplasm or the nucleus. Clp1p-GFP-NES cle events by dephosphorylating CDK sites. Their cell is absent from the nucleolus, the site where Clp1p is cycle-dependent changes in localization may be imthought to be sequestered and inactive. Surprisingly, portant to carry out distinct functions. Work in budthis does not affect cell-cycle progression as was reding and fission yeast suggested that Cdc14-like ported for Clp1p overexpression. Overproduction of phosphatases are inhibited by nucleolar sequestra-Clp1p leads to a delay in interphase, and this delay tion [1-3]. In S. cerevisiae, Cdc14p is released from causes cells to grow longer and septate at an increased the nucleolus by the FEAR network and Cdk1 [1, 4], size. Conversely, clp1⌬ mutants enter mitosis precowhereas the S. pombe CDC14-like phosphatase Clp1p ciously and divide as smaller cells than wild-type [2, 3]. (also known as Flp1p) is released at mitotic entry by Neither forcing Clp1p into or out of the nucleus had an unknown mechanism. The mitotic exit network a significant effect on the cell cycle during normal (MEN) in S. cerevisiae and its homologous network, growth, as judged by the cell size at cell division (Figthe septation initiation network (SIN), in S. pombe ure S2). act through an unknown mechanism to keep the In order to characterize the dynamics of Clp1p-GFPphosphatase out of the nucleolus in late mitosis [1]. NES and Clp1p-GFP-NLS localization, cultures were SIN-dependent cytoplasmic maintenance of Clp1p is synchronized in G2 phase with the cdc25-22 temperthought to be essential for the cytokinesis checkature-sensitive allele, then released from the block, and point, which blocks further rounds of nuclear division the localization pattern was analyzed in 10 min intervals until cytokinesis is completed. By targeting Clp1p to in fixed cells. Clp1p-GFP-NES localized at the SPB in the nucleus or the cytoplasm, we demonstrate discells arrested in G2 phase (Figure 2B a). Twenty minutes tinct functions for these pools of Clp1p in chromoafter release of the culture from the G2 arrest, Clp1psome segregation and cytokinesis, respectively. Our GFP-NES left the SPB and appeared in punctate dots results further suggest that the SIN does not keep at the medial cell cortex proximal to the nucleus, where Clp1p out of the nucleolus by regulating nucleolar afthe actin ring forms. As cells progressed into anaphase, finity, as proposed for S. cerevisiae Cdc14p, but inthe punctate Clp1p-GFP-NES signal assembled into a stead, Clp1p may be regulated by nuclear import/ concise ring, as previously reported for other ring comexport. ponents (Figures 2A and 2B b) [6]. After completion of ring constriction, Clp1p disappeared from the cell mid-Results and Discussion dle and reappeared at the SPBs (Figures 2A and 2B c). Clp1p-GFP-NLS was exclusively nucleolar in cells ar-Localization of Clp1p-NLS-GFP and Clp1p-NES-GFP rested in G2 phase (Figure 2D a) and appeared at ad-In order to distinguish between Clp1p nuclear and cytoditional nuclear spots, presumably the kinetochores, plasmic functions, we sought to target Clp1p either inshortly after release into mitosis. This coincided with side or outside the nucleus by tagging clp1-GFP with release of Clp1p-GFP-NLS throughout the nucleus in nuclear export signals (NES) or with nuclear localizaearly mitosis (Figure 2D b). As cells entered anaphase, tion signals (NLS). The resulting strains carry a single Clp1p-GFP-NLS localized to the mitotic spindle until tagged copy of clp1, expressed from its own promoter. spindle breakdown (Figure 2D c). Thereafter, Clp1p-The control Clp1p-GFP localized as expected to the GFP-NLS relocalized to the nucleolus (Figure 2D d). spindle pole body (SPB) and nucleolus in interphase In S. pombe, kinetochores are positioned in close and to the actin ring, the mitotic spindle, and kinetproximity to the SPBs in interphase and anaphase [7]. ochores during mitosis (Figures 1A and 1D) [2, 3, 5]. Therefore, SPB and kinetochore localization cannot be Clp1p-GFP-NES and Clp1p-GFP-NLS localized as predistinguished easily when proteins such as Clp1p localdicted: Clp1p-GFP-NES was found at cytoplasmic sites ize to both sites. However, the kinetochores are within such as the SPB during interphase and the contractile the nucleus and the SPBs outside the nucleus in inactin ring in mitosis (Figure 1B). Conversely, Clp1pterphase and at the end of anaphase [8]. Therefore, the GFP-NES was absent from nuclear sites such as the nuclear clp1-GFP-NLS and cytoplasmic clp1-GFP-NES nucleolus in interphase and the spindle during mitosis alleles allowed us to differentiate between Clp1p SPB and was only rarely detected at the kinetochores in and kinetochore localization. Because Clp1p-GFP-NLS some cells blocked in metaphase (Figures 1B and 1E). did not localize at a spot on the nuclear periphery during interphase, we concluded that it is absent from the kinetochores during interphase. Conversely, Clp1p-GFP

Molecular Biology of the Cell, Dec 1, 2012

In Schizosaccharomyces pombe, a late mitotic kinase pathway called the septation initiation netwo... more In Schizosaccharomyces pombe, a late mitotic kinase pathway called the septation initiation network (SIN) triggers cytokinesis. Here we show that the SIN is also involved in regulating anaphase spindle elongation and telophase nuclear positioning via inhibition of Klp2, a minus end-directed kinesin-14. Klp2 is known to localize to microtubules (MTs) and have roles in interphase nuclear positioning, mitotic chromosome alignment, and nuclear migration during karyogamy (nuclear fusion during mating). We observe SIN-dependent disappearance of Klp2 from MTs in anaphase, and we find that this is mediated by direct phosphorylation of Klp2 by the SIN kinase Sid2, which abrogates loading of Klp2 onto MTs by inhibiting its interaction with Mal3 (EB1 homologue). Disruption of Klp2 MT localization is required for efficient anaphase spindle elongation. Furthermore, when cytokinesis is delayed, SIN inhibition of Klp2 acts in concert with microtubules emanating from the equatorial microtubule-organizing center to position the nuclei away from the cell division site. These results reveal novel functions of the SIN in regulating the MT cytoskeleton and suggest that the SIN may have broader functions in regulating cellular organization in late mitosis than previously realized.

Molecular & Cellular Proteomics, Aug 1, 2012

Determining the localization, binding partners, and secondary modifications of individual protein... more Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions.

Developmental Cell, Dec 1, 2002

while nuclear division is unaffected, resulting in multinucleate cells. The SIN is a spindle pole... more while nuclear division is unaffected, resulting in multinucleate cells. The SIN is a spindle pole body (SPB)-associated GTPase-regulated kinase cascade. Two proteins,

Current Biology, Apr 1, 2002

Faithful actomyosin ring assembly is pivotal for successful cell division. The mechanisms by whic... more Faithful actomyosin ring assembly is pivotal for successful cell division. The mechanisms by which the actomyosin ring is assembled at the correct time and place remain unclear. Recent studies in fission yeast have shown that a myosin II-containing spot may be a novel progenitor structure essential for actomyosin ring assembly.

Cell Division, 2011

Regulation of cytoskeletal remodeling is essential for cell cycle transitions. In fission yeast t... more Regulation of cytoskeletal remodeling is essential for cell cycle transitions. In fission yeast two NDR kinase signaling cascades, MOR and SIN, regulate the actin cytoskeleton to promote polarized growth during interphase and cytokinesis respectively. Our understanding of how these signaling pathways are coordinated to assist transition between the two cell-cycle stages is limited. Here, we review work from our laboratory, which reveals that cross talk between the SIN and MOR pathways is required for inhibition of interphase polarity programs during cytokinesis. Given the conservation of NDR kinase signaling pathways, our results may define general mechanisms by which these pathways are coordinated in higher organisms.

Current Biology, Nov 1, 2004

The central spindle plays a key role in cytokinesis. Recent studies have shed new light on how as... more The central spindle plays a key role in cytokinesis. Recent studies have shed new light on how assembly of the central spindle is regulated, and also support a role for both the central spindle and astral microtubules in cytokinesis in animal cells. How cells determine the plane of cell division is one of the longest studied and most debated questions of cell biology (see [1] for review). Classic experiments by Ray Rappaport [2] on echinoderm embryos suggested that overlapping astral microtubules, emanating from the spindle poles, transmit a signal to the cell cortex to initiate furrowing. Other experiments, however, suggested that the signal to initiate furrowing comes from a structure called the spindle midzone, or the central spindle, particularly in smaller cells. A wide variety of more recent evidence strongly implicates the central spindle in cytokinesis. I will focus here largely on the central spindle, but will also discuss recent experiments that revive a role for astral microtubules in cytokinesis. When chromosomes begin moving to the spindle poles along kinetochore microtubules, the central spindle forms as a dense bundle of antiparallel nonkinetochore microtubules in the middle of the spindle. A number of proteins have been identified where loss of function disrupts both central spindle organization and cytokinesis. These include: the chromosomal passenger proteins INCENP, survivin, Aurora B kinase, and Borealin, the microtubule bundling protein PRC1, the kinesin MKLP1, and its associated Rho family GAP CYK-4 (reviewed in [3,4]). The various aliases of these proteins in different organisms are given in Table 1. Several new papers report results which have begun to shed light on how the timing of central spindle assembly is regulated. Earlier studies showed that formation of the central spindle is regulated by cyclin-dependent kinase (Cdk) activity. The central spindle begins to form at anaphase onset, when Cdk activity drops due to cyclin destruction. In fact expression of a nondestructible cyclin blocks central spindle formation and localization of Aurora B to the spindle, even though anaphase initiates normally [5,6]. Cdk regulation of central spindle formation seems to be mediated at least in part by direct phosphorylation of central spindle components (Figure 1). It has previously been shown that, in mammalian cells, the microtubule bundling protein PRC1 is a target of Cdk1 [7], and that Cdk phosphorylation seems to

Current Biology, Oct 1, 2002

The Cdc14 phosphatase was identified by its requirement for mitotic exit in budding yeast. Cdc14 ... more The Cdc14 phosphatase was identified by its requirement for mitotic exit in budding yeast. Cdc14 homologs exist throughout the eukaryotic kingdom, but it was unclear whether their function would also be conserved. Recent analyses in fission yeast, humans and now C. elegans suggest numerous other functions for this family of proteins.

Methods in molecular biology, 2014

Knowledge of an individual protein&am... more Knowledge of an individual protein's modifications, binding partners, and localization is essential for understanding complex biological networks. We recently described a fluorescent protein-based (mVenus) multifunctional affinity purification (MAP) tag that can be used both to purify a given protein and determine its localization (Ma et al., Mol Cell Proteomics 11:501-511, 2012). MAP purified protein complexes can be further analyzed to identify binding partners and posttranslational modifications by LC-MS/MS. The MAP approach offers rapid FACS-selection of stable clonal cell lines based on the expression level/fluorescence of the MAP-protein fusion. The MAP tag is highly efficient and shows little variability between proteins. Here we describe the general MAP purification method in detail, and show how it can be applied to a specific protein using the human Cdc14B phosphatase as an example.

FEBS Letters, May 28, 1999

In recent years, the actin cytoskeleton in Schizosaccharomyces pombe has become the subject of in... more In recent years, the actin cytoskeleton in Schizosaccharomyces pombe has become the subject of intense scrutiny. However, to date, only a single actin mutation has been identified. Described here is the isolation and characterization of four new cold-sensitive actin mutations. Sequence analysis of the mutant actin genes indicated that each of these mutations caused alterations in single amino acids that are conserved in all actin sequences. These mutants differ in their phenotypes. One of these mutations (act1-48) was identified as an extragenic suppressor of a mutation in the cdc4 gene, which is required for actin ring formation and cytokinesis. Interestingly, when act1-48 mutant cells were shifted to the restrictive temperature, actin patches were not detected but the actin ring formation and stability was unaffected. The three other mutations, act1-16, act1-32 and act1-67, primarily affected the actin ring formation or stability while F-actin patches did not seem to be substantially different in appearance. Given that the ultrastructural architectures of F-actin patches and the F-actin ring are presently unclear, these mutations, which affect one structure or the other, should be useful for future studies on the role of actin itself in the function of these F-actin-containing structures in S. pombe.

Journal of Biological Chemistry, Jun 1, 1999

Proper coordination of cytokinesis with chromosome separation during mitosis is crucial to ensure... more Proper coordination of cytokinesis with chromosome separation during mitosis is crucial to ensure that each daughter cell inherits an equivalent set of chromosomes. It has been proposed that one mechanism by which this is achieved is through temporally regulated myosin regulatory light chain (RLC) phosphorylation (Satterwhite, L. L., and Pollard, T. D. (1992) Curr. Opin. Cell Biol. 4, 43-52). A variety of evidence is consistent with this model. A direct test of the importance of RLC phosphorylation in vivo has been done only in Dictyostelium and Drosophila; phosphorylation of the RLC is essential in Drosophila (

The EMBO Journal, Dec 1, 1996

Profilins bind to monomeric actin and also interact with ligands such as phosphoinositide 4,5-bis... more Profilins bind to monomeric actin and also interact with ligands such as phosphoinositide 4,5-bisphosphate, the proline-rich protein VASP and a complex of four to six polypeptides identified in Acanthamoeba that includes two actin-related proteins. Here, we report the identification and characterization of an essential gene from Schizosaccharomyces pombe, sop2 +, a mutation in which rescues the temperature-sensitive lethality of a profilin mutation, cdc3-124. The sop2-1 mutant is defective for cell elongation and septation, suggesting that it is involved in multiple cortical actin-requiring processes. Consistent with a role in actin cytoskeletal function, negative interactions have been identified between sop2-1 and actl-48, a mutant allele of actin. Sop2p is a novel 377 amino acid polypeptide with similarity to proteins of the 3-transducin repeat family. Sop2p-related proteins have been identified by sequencing projects in diverse species, and we have isolated a human cDNA highly related to sop2 +, SOP2 Hs, which functionally complements the sop2-1 mutation. Sop2p proteins from all species contain peptide sequences identical or highly similar to two peptide sequences from an Acanthamoeba ,B-transducin repeat protein present in the profilin binding complex. Biochemical analyses demonstrate that Sop2p is present in a complex which also contains the actin-related protein, Arp3p. Immunofluorescence studies reveal the presence of Sop2p in (i) punctate structures distributed throughout the cell, (ii) cables that extend the length of the cell, and (iii) a medial band in a small percentage of septating cells. Collectively these data demonstrate the interaction of Sop2p with Arp3p, profilin and actin.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Sep 11, 2008

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Jan 23, 2009

bioRxiv (Cold Spring Harbor Laboratory), Oct 12, 2023

A key step in regulation of Hippo pathway signaling in response to mechanical tension is recruitm... more A key step in regulation of Hippo pathway signaling in response to mechanical tension is recruitment of the LIM domain proteins TRIP6 and LIMD1 to adherens junctions. Mechanical tension also triggers TRIP6 and LIMD1 to bind and inhibit the Hippo pathway kinase LATS1. How TRIP6 and LIMD1 are recruited to adherens junctions in response to tension is not clear, but previous studies suggested that they could be regulated by the known mechanosensory proteins a-catenin and vinculin at adherens junctions. We found that the three LIM domains of TRIP6 and LIMD1 are necessary and sufficient for tension-dependent localization to adherens junctions. The LIM domains of TRIP6, LIMD1, and certain other LIM domain proteins have been shown to bind to actin networks under strain/tension. Consistent with this, we show that TRIP6 and LIMD1 colocalize with the ends of actin fibers at adherens junctions. Point mutations in a key conserved residue in each LIM domain that are predicted to impair binding to f-actin under strain inhibits TRIP6 and LIMD1 localization to adherens junctions and their ability to bind to and recruit LATS1 to adherens junctions. Together these results show that the ability of TRIP6 and LIMD1 to bind to strained actin underlies their ability to localize to adherens junctions and regulate LATS1 in response to mechanical tension.

Journal of Cell Science, 2003

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Nov 10, 2008

Cytokinesis is powered by the contraction of actomyosin filaments within the newly assembled cont... more Cytokinesis is powered by the contraction of actomyosin filaments within the newly assembled contractile ring. Microtubules are a spindle component that is essential for the induction of cytokinesis. This induction could use central spindle and/or astral microtubules to stimulate cortical contraction around the spindle equator (equatorial stimulation). Alternatively, or in addition, induction could rely on astral microtubules to relax the polar cortex (polar relaxation). To investigate the relationship between microtubules, cortical stiffness, and contractile ring assembly, we used different configurations of microtubules to manipulate the distribution of actin in living silkworm spermatocytes. Mechanically repositioned, noninterdigitating microtubules can induce redistribution of actin at any region of the cortex by locally excluding cortical actin filaments. This cortical flow of actin promotes regional relaxation while increasing tension elsewhere (normally at the equatorial cortex). In contrast, repositioned interdigitating microtubule bundles use a novel mechanism to induce local stimulation of contractility anywhere within the cortex; at the antiparallel plus ends of central spindle microtubules, actin aggregates are rapidly assembled de novo and transported laterally to the equatorial cortex. Relaxation depends on microtubule dynamics but not on RhoA activity, whereas stimulation depends on RhoA activity but is largely independent of microtubule dynamics. We conclude that polar relaxation and equatorial stimulation mechanisms redundantly supply actin for contractile ring assembly, thus increasing the fidelity of cleavage.

Springer eBooks, 2013

S-adenosylmethionine (Fig. 1) is synthesized from ATP and L-methionine and acts as a donor of met... more S-adenosylmethionine (Fig. 1) is synthesized from ATP and L-methionine and acts as a donor of methyl groups in prokaryote as well as eukaryote. By the transfer of a methyl group, S-adenosylmethionine is converted into S-adenosylhomocysteine, which is broken down to homocysteine and adenosine. Homocysteine is then converted to methionine. S-adenosylmethionine is also used for synthesis of cysteine as well as polyamines.

Current Biology, Jun 1, 2010

simply, we might simply double the term for the relative siring success of males compared with he... more simply, we might simply double the term for the relative siring success of males compared with hermaphrodites (Figure 2). The previous implausibility of androdioecy in P. angustifolia was thus not due to the unavailability of an appropriate theoretical framework, but rather to a violation of the simple implicit assumption that all hermaphrodites are potential sires for all progeny. The genetic details exposed by Saumitou-Laprade et al.'s study [10] are fascinating and unexpected, but androdioecy in P. angustifolia can be explained by the old paradigm after all, which simply compares the expected relative siring prospects of different sexual phenotypes.

Current Biology, Aug 1, 2005

Medical Center lus in interphase and the spindle during anaphase (Figure 1C) as well as the kinet... more Medical Center lus in interphase and the spindle during anaphase (Figure 1C) as well as the kinetochores in metaphase Worcester, Massachusetts 01605 (Figure 1F; see Figure S1 in the Supplemental Data available with this article online). Both Clp1p-GFP-NES and Summary Clp1p-GFP-NLS presumably still shuttle across the nuclear envelope, but their steady-state levels are shifted Cdc14-like phosphatases regulate a variety of cell cyto either the cytoplasm or the nucleus. Clp1p-GFP-NES cle events by dephosphorylating CDK sites. Their cell is absent from the nucleolus, the site where Clp1p is cycle-dependent changes in localization may be imthought to be sequestered and inactive. Surprisingly, portant to carry out distinct functions. Work in budthis does not affect cell-cycle progression as was reding and fission yeast suggested that Cdc14-like ported for Clp1p overexpression. Overproduction of phosphatases are inhibited by nucleolar sequestra-Clp1p leads to a delay in interphase, and this delay tion [1-3]. In S. cerevisiae, Cdc14p is released from causes cells to grow longer and septate at an increased the nucleolus by the FEAR network and Cdk1 [1, 4], size. Conversely, clp1⌬ mutants enter mitosis precowhereas the S. pombe CDC14-like phosphatase Clp1p ciously and divide as smaller cells than wild-type [2, 3]. (also known as Flp1p) is released at mitotic entry by Neither forcing Clp1p into or out of the nucleus had an unknown mechanism. The mitotic exit network a significant effect on the cell cycle during normal (MEN) in S. cerevisiae and its homologous network, growth, as judged by the cell size at cell division (Figthe septation initiation network (SIN), in S. pombe ure S2). act through an unknown mechanism to keep the In order to characterize the dynamics of Clp1p-GFPphosphatase out of the nucleolus in late mitosis [1]. NES and Clp1p-GFP-NLS localization, cultures were SIN-dependent cytoplasmic maintenance of Clp1p is synchronized in G2 phase with the cdc25-22 temperthought to be essential for the cytokinesis checkature-sensitive allele, then released from the block, and point, which blocks further rounds of nuclear division the localization pattern was analyzed in 10 min intervals until cytokinesis is completed. By targeting Clp1p to in fixed cells. Clp1p-GFP-NES localized at the SPB in the nucleus or the cytoplasm, we demonstrate discells arrested in G2 phase (Figure 2B a). Twenty minutes tinct functions for these pools of Clp1p in chromoafter release of the culture from the G2 arrest, Clp1psome segregation and cytokinesis, respectively. Our GFP-NES left the SPB and appeared in punctate dots results further suggest that the SIN does not keep at the medial cell cortex proximal to the nucleus, where Clp1p out of the nucleolus by regulating nucleolar afthe actin ring forms. As cells progressed into anaphase, finity, as proposed for S. cerevisiae Cdc14p, but inthe punctate Clp1p-GFP-NES signal assembled into a stead, Clp1p may be regulated by nuclear import/ concise ring, as previously reported for other ring comexport. ponents (Figures 2A and 2B b) [6]. After completion of ring constriction, Clp1p disappeared from the cell mid-Results and Discussion dle and reappeared at the SPBs (Figures 2A and 2B c). Clp1p-GFP-NLS was exclusively nucleolar in cells ar-Localization of Clp1p-NLS-GFP and Clp1p-NES-GFP rested in G2 phase (Figure 2D a) and appeared at ad-In order to distinguish between Clp1p nuclear and cytoditional nuclear spots, presumably the kinetochores, plasmic functions, we sought to target Clp1p either inshortly after release into mitosis. This coincided with side or outside the nucleus by tagging clp1-GFP with release of Clp1p-GFP-NLS throughout the nucleus in nuclear export signals (NES) or with nuclear localizaearly mitosis (Figure 2D b). As cells entered anaphase, tion signals (NLS). The resulting strains carry a single Clp1p-GFP-NLS localized to the mitotic spindle until tagged copy of clp1, expressed from its own promoter. spindle breakdown (Figure 2D c). Thereafter, Clp1p-The control Clp1p-GFP localized as expected to the GFP-NLS relocalized to the nucleolus (Figure 2D d). spindle pole body (SPB) and nucleolus in interphase In S. pombe, kinetochores are positioned in close and to the actin ring, the mitotic spindle, and kinetproximity to the SPBs in interphase and anaphase [7]. ochores during mitosis (Figures 1A and 1D) [2, 3, 5]. Therefore, SPB and kinetochore localization cannot be Clp1p-GFP-NES and Clp1p-GFP-NLS localized as predistinguished easily when proteins such as Clp1p localdicted: Clp1p-GFP-NES was found at cytoplasmic sites ize to both sites. However, the kinetochores are within such as the SPB during interphase and the contractile the nucleus and the SPBs outside the nucleus in inactin ring in mitosis (Figure 1B). Conversely, Clp1pterphase and at the end of anaphase [8]. Therefore, the GFP-NES was absent from nuclear sites such as the nuclear clp1-GFP-NLS and cytoplasmic clp1-GFP-NES nucleolus in interphase and the spindle during mitosis alleles allowed us to differentiate between Clp1p SPB and was only rarely detected at the kinetochores in and kinetochore localization. Because Clp1p-GFP-NLS some cells blocked in metaphase (Figures 1B and 1E). did not localize at a spot on the nuclear periphery during interphase, we concluded that it is absent from the kinetochores during interphase. Conversely, Clp1p-GFP

Molecular Biology of the Cell, Dec 1, 2012

In Schizosaccharomyces pombe, a late mitotic kinase pathway called the septation initiation netwo... more In Schizosaccharomyces pombe, a late mitotic kinase pathway called the septation initiation network (SIN) triggers cytokinesis. Here we show that the SIN is also involved in regulating anaphase spindle elongation and telophase nuclear positioning via inhibition of Klp2, a minus end-directed kinesin-14. Klp2 is known to localize to microtubules (MTs) and have roles in interphase nuclear positioning, mitotic chromosome alignment, and nuclear migration during karyogamy (nuclear fusion during mating). We observe SIN-dependent disappearance of Klp2 from MTs in anaphase, and we find that this is mediated by direct phosphorylation of Klp2 by the SIN kinase Sid2, which abrogates loading of Klp2 onto MTs by inhibiting its interaction with Mal3 (EB1 homologue). Disruption of Klp2 MT localization is required for efficient anaphase spindle elongation. Furthermore, when cytokinesis is delayed, SIN inhibition of Klp2 acts in concert with microtubules emanating from the equatorial microtubule-organizing center to position the nuclei away from the cell division site. These results reveal novel functions of the SIN in regulating the MT cytoskeleton and suggest that the SIN may have broader functions in regulating cellular organization in late mitosis than previously realized.

Molecular & Cellular Proteomics, Aug 1, 2012

Determining the localization, binding partners, and secondary modifications of individual protein... more Determining the localization, binding partners, and secondary modifications of individual proteins is crucial for understanding protein function. Several tags have been constructed for protein localization or purification under either native or denaturing conditions, but few tags permit all three simultaneously. Here, we describe a multifunctional tandem affinity purification (MAP) method that is both highly efficient and enables protein visualization. The MAP tag utilizes affinity tags inserted into an exposed surface loop of mVenus offering two advantages: (1) mVenus fluorescence can be used for protein localization or FACS-based selection of cell lines; and (2) spatial separation of the affinity tags from the protein results in high recovery and reduced variability between proteins. MAP purification was highly efficient in multiple organisms for all proteins tested. As a test case, MAP combined with liquid chromatography-tandem MS identified known and new candidate binding partners and modifications of the kinase Plk1. Thus the MAP tag is a new powerful tool for determining protein modification, localization, and interactions.

Developmental Cell, Dec 1, 2002

while nuclear division is unaffected, resulting in multinucleate cells. The SIN is a spindle pole... more while nuclear division is unaffected, resulting in multinucleate cells. The SIN is a spindle pole body (SPB)-associated GTPase-regulated kinase cascade. Two proteins,

Current Biology, Apr 1, 2002

Faithful actomyosin ring assembly is pivotal for successful cell division. The mechanisms by whic... more Faithful actomyosin ring assembly is pivotal for successful cell division. The mechanisms by which the actomyosin ring is assembled at the correct time and place remain unclear. Recent studies in fission yeast have shown that a myosin II-containing spot may be a novel progenitor structure essential for actomyosin ring assembly.

Cell Division, 2011

Regulation of cytoskeletal remodeling is essential for cell cycle transitions. In fission yeast t... more Regulation of cytoskeletal remodeling is essential for cell cycle transitions. In fission yeast two NDR kinase signaling cascades, MOR and SIN, regulate the actin cytoskeleton to promote polarized growth during interphase and cytokinesis respectively. Our understanding of how these signaling pathways are coordinated to assist transition between the two cell-cycle stages is limited. Here, we review work from our laboratory, which reveals that cross talk between the SIN and MOR pathways is required for inhibition of interphase polarity programs during cytokinesis. Given the conservation of NDR kinase signaling pathways, our results may define general mechanisms by which these pathways are coordinated in higher organisms.

Current Biology, Nov 1, 2004

The central spindle plays a key role in cytokinesis. Recent studies have shed new light on how as... more The central spindle plays a key role in cytokinesis. Recent studies have shed new light on how assembly of the central spindle is regulated, and also support a role for both the central spindle and astral microtubules in cytokinesis in animal cells. How cells determine the plane of cell division is one of the longest studied and most debated questions of cell biology (see [1] for review). Classic experiments by Ray Rappaport [2] on echinoderm embryos suggested that overlapping astral microtubules, emanating from the spindle poles, transmit a signal to the cell cortex to initiate furrowing. Other experiments, however, suggested that the signal to initiate furrowing comes from a structure called the spindle midzone, or the central spindle, particularly in smaller cells. A wide variety of more recent evidence strongly implicates the central spindle in cytokinesis. I will focus here largely on the central spindle, but will also discuss recent experiments that revive a role for astral microtubules in cytokinesis. When chromosomes begin moving to the spindle poles along kinetochore microtubules, the central spindle forms as a dense bundle of antiparallel nonkinetochore microtubules in the middle of the spindle. A number of proteins have been identified where loss of function disrupts both central spindle organization and cytokinesis. These include: the chromosomal passenger proteins INCENP, survivin, Aurora B kinase, and Borealin, the microtubule bundling protein PRC1, the kinesin MKLP1, and its associated Rho family GAP CYK-4 (reviewed in [3,4]). The various aliases of these proteins in different organisms are given in Table 1. Several new papers report results which have begun to shed light on how the timing of central spindle assembly is regulated. Earlier studies showed that formation of the central spindle is regulated by cyclin-dependent kinase (Cdk) activity. The central spindle begins to form at anaphase onset, when Cdk activity drops due to cyclin destruction. In fact expression of a nondestructible cyclin blocks central spindle formation and localization of Aurora B to the spindle, even though anaphase initiates normally [5,6]. Cdk regulation of central spindle formation seems to be mediated at least in part by direct phosphorylation of central spindle components (Figure 1). It has previously been shown that, in mammalian cells, the microtubule bundling protein PRC1 is a target of Cdk1 [7], and that Cdk phosphorylation seems to

Current Biology, Oct 1, 2002

The Cdc14 phosphatase was identified by its requirement for mitotic exit in budding yeast. Cdc14 ... more The Cdc14 phosphatase was identified by its requirement for mitotic exit in budding yeast. Cdc14 homologs exist throughout the eukaryotic kingdom, but it was unclear whether their function would also be conserved. Recent analyses in fission yeast, humans and now C. elegans suggest numerous other functions for this family of proteins.

Methods in molecular biology, 2014

Knowledge of an individual protein&am... more Knowledge of an individual protein's modifications, binding partners, and localization is essential for understanding complex biological networks. We recently described a fluorescent protein-based (mVenus) multifunctional affinity purification (MAP) tag that can be used both to purify a given protein and determine its localization (Ma et al., Mol Cell Proteomics 11:501-511, 2012). MAP purified protein complexes can be further analyzed to identify binding partners and posttranslational modifications by LC-MS/MS. The MAP approach offers rapid FACS-selection of stable clonal cell lines based on the expression level/fluorescence of the MAP-protein fusion. The MAP tag is highly efficient and shows little variability between proteins. Here we describe the general MAP purification method in detail, and show how it can be applied to a specific protein using the human Cdc14B phosphatase as an example.

FEBS Letters, May 28, 1999

In recent years, the actin cytoskeleton in Schizosaccharomyces pombe has become the subject of in... more In recent years, the actin cytoskeleton in Schizosaccharomyces pombe has become the subject of intense scrutiny. However, to date, only a single actin mutation has been identified. Described here is the isolation and characterization of four new cold-sensitive actin mutations. Sequence analysis of the mutant actin genes indicated that each of these mutations caused alterations in single amino acids that are conserved in all actin sequences. These mutants differ in their phenotypes. One of these mutations (act1-48) was identified as an extragenic suppressor of a mutation in the cdc4 gene, which is required for actin ring formation and cytokinesis. Interestingly, when act1-48 mutant cells were shifted to the restrictive temperature, actin patches were not detected but the actin ring formation and stability was unaffected. The three other mutations, act1-16, act1-32 and act1-67, primarily affected the actin ring formation or stability while F-actin patches did not seem to be substantially different in appearance. Given that the ultrastructural architectures of F-actin patches and the F-actin ring are presently unclear, these mutations, which affect one structure or the other, should be useful for future studies on the role of actin itself in the function of these F-actin-containing structures in S. pombe.

Journal of Biological Chemistry, Jun 1, 1999

Proper coordination of cytokinesis with chromosome separation during mitosis is crucial to ensure... more Proper coordination of cytokinesis with chromosome separation during mitosis is crucial to ensure that each daughter cell inherits an equivalent set of chromosomes. It has been proposed that one mechanism by which this is achieved is through temporally regulated myosin regulatory light chain (RLC) phosphorylation (Satterwhite, L. L., and Pollard, T. D. (1992) Curr. Opin. Cell Biol. 4, 43-52). A variety of evidence is consistent with this model. A direct test of the importance of RLC phosphorylation in vivo has been done only in Dictyostelium and Drosophila; phosphorylation of the RLC is essential in Drosophila (

The EMBO Journal, Dec 1, 1996

Profilins bind to monomeric actin and also interact with ligands such as phosphoinositide 4,5-bis... more Profilins bind to monomeric actin and also interact with ligands such as phosphoinositide 4,5-bisphosphate, the proline-rich protein VASP and a complex of four to six polypeptides identified in Acanthamoeba that includes two actin-related proteins. Here, we report the identification and characterization of an essential gene from Schizosaccharomyces pombe, sop2 +, a mutation in which rescues the temperature-sensitive lethality of a profilin mutation, cdc3-124. The sop2-1 mutant is defective for cell elongation and septation, suggesting that it is involved in multiple cortical actin-requiring processes. Consistent with a role in actin cytoskeletal function, negative interactions have been identified between sop2-1 and actl-48, a mutant allele of actin. Sop2p is a novel 377 amino acid polypeptide with similarity to proteins of the 3-transducin repeat family. Sop2p-related proteins have been identified by sequencing projects in diverse species, and we have isolated a human cDNA highly related to sop2 +, SOP2 Hs, which functionally complements the sop2-1 mutation. Sop2p proteins from all species contain peptide sequences identical or highly similar to two peptide sequences from an Acanthamoeba ,B-transducin repeat protein present in the profilin binding complex. Biochemical analyses demonstrate that Sop2p is present in a complex which also contains the actin-related protein, Arp3p. Immunofluorescence studies reveal the presence of Sop2p in (i) punctate structures distributed throughout the cell, (ii) cables that extend the length of the cell, and (iii) a medial band in a small percentage of septating cells. Collectively these data demonstrate the interaction of Sop2p with Arp3p, profilin and actin.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Sep 11, 2008

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Jan 23, 2009

bioRxiv (Cold Spring Harbor Laboratory), Oct 12, 2023

A key step in regulation of Hippo pathway signaling in response to mechanical tension is recruitm... more A key step in regulation of Hippo pathway signaling in response to mechanical tension is recruitment of the LIM domain proteins TRIP6 and LIMD1 to adherens junctions. Mechanical tension also triggers TRIP6 and LIMD1 to bind and inhibit the Hippo pathway kinase LATS1. How TRIP6 and LIMD1 are recruited to adherens junctions in response to tension is not clear, but previous studies suggested that they could be regulated by the known mechanosensory proteins a-catenin and vinculin at adherens junctions. We found that the three LIM domains of TRIP6 and LIMD1 are necessary and sufficient for tension-dependent localization to adherens junctions. The LIM domains of TRIP6, LIMD1, and certain other LIM domain proteins have been shown to bind to actin networks under strain/tension. Consistent with this, we show that TRIP6 and LIMD1 colocalize with the ends of actin fibers at adherens junctions. Point mutations in a key conserved residue in each LIM domain that are predicted to impair binding to f-actin under strain inhibits TRIP6 and LIMD1 localization to adherens junctions and their ability to bind to and recruit LATS1 to adherens junctions. Together these results show that the ability of TRIP6 and LIMD1 to bind to strained actin underlies their ability to localize to adherens junctions and regulate LATS1 in response to mechanical tension.

Journal of Cell Science, 2003