Cell and Developmental Biology Commons^™

Growth Factor–Induced Shedding Of Syndecan-1 Confers Glypican-1 Dependence On Mitogenic Responses Of Cancer Cells, Kan Ding, Martha Lopez-Burks, José A. Sánchez-Duran, Murray Korc, Arthur D. Lander

Dartmouth Scholarship

The cell surface heparan sulfate proteoglycan (HSPG) glypican-1 is up-regulated by pancreatic and breast cancer cells, and its removal renders such cells insensitive to many growth factors. We sought to explain why the cell surface HSPG syndecan-1, which is also up-regulated by these cells and is a known growth factor coreceptor, does not compensate for glypican-1 loss. We show that the initial responses of these cells to the growth factor FGF2 are not glypican dependent, but they become so over time as FGF2 induces shedding of syndecan-1. Manipulations that retain syndecan-1 on the cell surface make long-term FGF2 responses glypican …

Calmodulin And Pf6 Are Components Of A Complex That Localizes To The C1 Microtubule Of The Flagellar Central Apparatus, Matthew J. Wargo, Erin E. Dymek, Elizabeth F. Smith

Dartmouth Scholarship

Studies of flagellar motility in Chlamydomonas mutants lacking specific central apparatus components have supported the hypothesis that the inherent asymmetry of this structure provides important spatial cues for asymmetric regulation of dynein activity. These studies have also suggested that specific projections associated with the C1 and C2 central tubules make unique contributions to modulating motility; yet, we still do not know the identities of most polypeptides associated with the central tubules. To identify components of the C1a projection, we took an immunoprecipitation approach using antibodies generated against PF6. The pf6 mutant lacks the C1a projection and possesses flagella that only …

Crystal Structure Of The Gtpase Domain Of Rat Dynamin 1, Thomas F. Reubold, Susanne Eschenburg, Andreas Becker, Marilyn Leonard, Sandra L. Schmid, Richard B. Vallee, F. Jon Kull, Dietmar J. Manstein

Dartmouth Scholarship

Here, we present the 1.9-A crystal structure of the nucleotide-free GTPase domain of dynamin 1 from Rattus norvegicus. The structure corresponds to an extended form of the canonical GTPase fold observed in Ras proteins. Both nucleotide-binding switch motifs are well resolved, adopting conformations that closely resemble a GTP-bound state not previously observed for nucleotide-free GTPases. Two highly conserved arginines, Arg-66 and Arg-67, greatly restrict the mobility of switch I and are ideally positioned to relay information about the nucleotide state to other parts of the protein. Our results support a model in which switch I residue Arg-59 gates GTP binding …

Arsenite Regulates Cystic Fibrosis Transmem­Brane Conductance Regulator And P-Glycoprotein: Evidence Of Pathway Independence, Rangan Maitra, Joshua Hamilton

Dartmouth Scholarship

In the past, people have argued for and against the theory of reciprocal regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and P-glycoprotein (Pgp). Data have indicated that this may occur in vitro during drug-induced selection of cells, and in vivo during development. Much of this debate has been caused by a severe lack of mechanistic details involved in such regulation. Our past data indicate that certain Pgp modulators can affect CFTR expression and function. The goal of this study was to investigate the effects of trivalent arsenic (arsenite), a known transcriptional activator of Pgp, on CFTR expression. In …

Kinetics And Relative Importance Of Phosphorolytic And Hydrolytic Cleavage Of Cellodextrins And Cellobiose In Cell Extracts Of Clostridium Thermocellum, Yie.-Heng P. Zhang, Lee R. Lynd

Dartmouth Scholarship

Rates of phosphorolytic cleavage of 􏰁-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temper- atures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by >20-fold …

The Caenorhabditis Elegans F-Box Protein Sel-10 Promotes Female Development And May Target Fem-1 And Fem-3 For Degradation By The Proteasome, Sibylle Jager, Hillel T. Schwartz, H. Robert Horvitz, Barbara Conradt

Dartmouth Scholarship

The Caenorhabditis elegans F-box protein SEL-10 and its human homolog have been proposed to regulate LIN-12 Notch signaling by targeting for ubiquitin-mediated proteasomal degradation LIN-12 Notch proteins and SEL-12 PS1 presenilins, the latter of which have been implicated in Alzheimer's disease. We found that sel-10 is the same gene as egl-41, which previously had been defined by gain-of-function mutations that semidominantly cause masculinization of the hermaphrodite soma. Our results demonstrate that mutations causing loss-of-function of sel-10 also have masculinizing activity, indicating that sel-10 functions to promote female development. Genetically, sel-10 acts upstream of the genes fem-1, fem-2, and fem-3 and …

Bcma Is Essential For The Survival Of Long-Lived Bone Marrow Plasma Cells, Brian P. O'Connor, Vanitha S. Raman, Loren D. Erickson, W. James Cook, Lehn K. Weaver, Cory Ahonen, Ling-Li Lin, George Mantchev, Richard J. Bram, Randolph J. Noelle

Dartmouth Scholarship

Long-lived humoral immunity is manifested by the ability of bone marrow plasma cells (PCs) to survive for extended periods of time. Recent studies have underscored the importance of BLyS and APRIL as factors that can support the survival of B lineage lymphocytes. We show that BLyS can sustain PC survival in vitro, and this survival can be further enhanced by inter- leukin 6. Selective up-regulation of Mcl-1 in PCs by BLyS suggests that this 􏰀-apoptotic gene product may play an important role in PC survival. Blockade of BLyS, via transmembrane activator and cyclophilin ligand interactor–immunoglobulin treatment, inhibited PC survival in …

Analysis Of Microtubule Sliding Patterns In Chlamydomonas Flagellar Axonemes Reveals Dynein Activity On Specific Doublet Microtubules, M. J. Wargo, Mark A. Mcpeek, Elizabeth F. Smith

Dartmouth Scholarship

Generating the complex waveforms characteristic of beating eukaryotic cilia and flagella requires spatial regulation of dynein-driven microtubule sliding. To generate bending, one prediction is that dynein arms alternate between active and inactive forms on specific subsets of doublet microtubules. Using an in vitro microtubule sliding assay combined with a structural approach, we determined that ATP induces sliding between specific subsets of doublet microtubules, apparently capturing one phase of the beat cycle. These studies were also conducted using high Ca2+ conditions. InChlamydomonas, high Ca2+ induces changes in waveform which are predicted to result from regulating dynein

activity on specific microtubules. Our …

A Role For Yip1p In Copii Vesicle Biogenesis, Matthew Heidtman, Catherine Z. Chen, Ruth N. Collins, Charles Barlowe

Dartmouth Scholarship

Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes …

Minus-End Capture Of Preformed Kinetochore Fibers Contributes To Spindle Morphogenesis, Alexey Khodjakov, Lily Copenagle, Michael B. Gordon, Duane A. Compton, Tarun M. Kapoor

Dartmouth Scholarship

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient …

Asymmetry Of The Central Apparatus Defines The Location Of Active Microtubule Sliding In Chlamydomonas Flagella, Matthew J. Wargo, Elizabeth F. Smith

Dartmouth Scholarship

Regulation of ciliary and flagellar motility requires spatial control of dynein-driven microtubule sliding. However, the mechanism for regulating the location and symmetry of dynein activity is not understood. One hypothesis is that the asymmetrically organized central apparatus, through interactions with the radial spokes, transmits a signal to regulate dynein-driven microtubule sliding between subsets of doublet microtubules. Based on this model, we hypothesized that the orientation of the central apparatus defines positions of active microtubule sliding required to control bending in the axoneme. To test this, we induced microtubule sliding in axonemes isolated from wild-type and mutant Chlamydomonas cells, and then …

Searching For The Middle Ground: Mechanisms Of Chromosome Alignment During Mitosis, Tarun M. Kapoor, Duane A. Compton

Dartmouth Scholarship

The contributions of key molecules predicted to align chromosomes at the center of the mitotic spindle have been recently examined. New results dictate that models for how chromosomes align during the early stages of mitosis must be revised to integrate properties of microtubule-based motor proteins as well as microtubule dynamics.

A Cycle Of Vam7p Release From And Ptdins 3-P–Dependent Rebinding To The Yeast Vacuole Is Required For Homotypic Vacuole Fusion, Christine Boeddinghaus, Alexey J. Merz, Rico Laage, Christian Ungermann

Dartmouth Scholarship

Vacuole fusion requires a coordinated cascade of priming, docking, and fusion. SNARE proteins have been implicated in the fusion itself, although their precise role in the cascade remains unclear. We now report that the vacuolar SNAP-23 homologue Vam7p is a mobile element of the SNARE complex, which moves from an initial association with the cis-SNARE complex via a soluble intermediate to the docking site. Soluble Vam7p is specifically recruited to vacuoles and can rescue a fusion reaction poisoned with antibodies to Vam7p. Both the recombinant Vam7p PX domain and a FYVE domain construct of human Hrs block the recruitment of …

Ube1l Is A Retinoid Target That Triggers Pml/Rarα Degradation And Apoptosis In Acute Promyelocytic Leukemia, Sutisak Kitareewan, Ian Pitha-Rowe, David Sekula, Christopher H. Lowrey, Michael J. Nemeth, Todd R. Golub, Sarah J. Freemantle, Ethan Dmitrovsky

Dartmouth Scholarship

All-trans-retinoic acid (RA) treatment induces remissions in acute promyelocytic leukemia (APL) cases expressing the t(15;17) product, promyelocytic leukemia (PML)/RA receptor α (RARα). Microarray analyses previously revealed induction of UBE1L (ubiquitin-activating enzyme E1-like) after RA treatment of NB4 APL cells. We report here that this occurs within 3 h in RA-sensitive but not RA-resistant APL cells, implicating UBE1L as a direct retinoid target. A 1.3-kb fragment of the UBE1L promoter was capable of mediating transcriptional response to RA in a retinoid receptor-selective manner. PML/RARα, a repressor of RA target genes, abolished this UBE1L promoter activity. A hallmark of …

Nuclear Export Of 60s Ribosomal Subunits Depends On Xpo1p And Requires A Nuclear Export Sequence-Containing Factor, Nmd3p, That Associates With The Large Subunit Protein Rpl10p, Olivier Gadal, Daniela Strau, Jacques Kessl, Bernard Trumpower

Dartmouth Scholarship

Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We …

Chromosome Movement In Mitosis Requires Microtubule Anchorage At Spindle Poles, Michael B. Gordon, Louisa Howard, Duane A. Compton

Dartmouth Scholarship

Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. …

Erv41p And Erv46p New Components Of Copii Vesicles Involved In Transport Between The Er And Golgi Complex, Stefan Otte, William J. Belden, Matthew Heidtman, Jay Liu, Ole N. Jensen, Charles Barlowe

Dartmouth Scholarship

Proteins contained on purified COPII vesicles were analyzed by matrix-assisted laser desorption ionization mass spectrometry combined with database searching. We identified four known vesicle proteins (Erv14p, Bet1p, Emp24p, and Erv25p) and an additional nine species (Yip3p, Rer1p, Erp1p, Erp2p, Erv29p, Yif1p, Erv41p, Erv46p, and Emp47p) that had not been localized to ER vesicles. Using antibodies, we demonstrate that these proteins are selectively and efficiently packaged into COPII vesicles. Three of the newly identified vesicle proteins (Erv29p, Erv41p, and Erv46p) represent uncharacterized integral membrane proteins that are conserved across species. Erv41p and Erv46p were further characterized. These proteins co- localized to …

Fast Transport Of Neurofilament Protein Along Microtubules In Squid Axoplasm, Veena Prahlad, Brian T. Helfand, George M. Langford, Ron D. Vale, Robert D. Goldman

Dartmouth Scholarship

Using squid axoplasm as a model system, we have visualized the fast transport of non-filamentous neurofilament protein particles along axonal microtubules. This transport occurs at speeds of 0.5-1.0 microm/second and the majority of neurofilament particles stain with kinesin antibody. These observations demonstrate, for the first time, that fast (0.5-1.0 microm/second) transport of neurofilament proteins occurs along microtubules. In addition, our studies suggest that neurofilament protein can be transported as non-membrane bound, nonfilamentous subunits along axons, and that the transport is kinesin-dependent. Microtubule-based fast transport might therefore provide a mechanism for the distribution and turnover of neurofilament, and perhaps other cytoskeletal …

The Docking Stage Of Yeast Vacuole Fusion Requires The Transfer Of Proteins From A Cis-Snare Complex To A Rab/Ypt Protein, Albert Price, Darren Seals, William Wickner, Christian Ungermann

Dartmouth Scholarship

The homotypic fusion of yeast vacuoles requires Sec18p (NSF)-driven priming to allow vacuole docking, but the mechanism that links priming and docking is unknown. We find that a large multisubunit protein called the Vam2/6p complex is bound to cis-paired SNAP receptors (SNAREs) on isolated vacuoles. This association of the Vam2/6p complex with the cis-SNARE complex is disrupted during priming. The Vam2/6p complex then binds to Ypt7p, a guanosine triphosphate binding protein of the Rab family, to initiate productive contact between vacuoles. Thus, cis-SNARE complexes can contain Rab/Ypt effectors, and these effectors can be mobilized by NSF/Sec18p-driven priming, allowing their direct …

The Kinesin-Related Protein, Hset, Opposes The Activity Of Eg5 And Cross-Links Microtubules In The Mammalian Mitotic Spindle, Vicki Mountain, Calvin Simerly, Louisa Howard, Asako Ando, Gerald Schatten, Duane A. Compton

Dartmouth Scholarship

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without …

Cell Cycle-Dependent Sequencing Of Cell Fate Decisions In Caenorhabditis Elegans Vulva Precursor Cells, Victor Ambros

Dartmouth Scholarship

In Caenorhabditis elegans, the fates of the six multipotent vulva precursor cells (VPCs) are specified by extracellular signals. One VPC expresses the primary (1°) fate in response to a Ras-mediated inductive signal from the gonad. The two VPCs flanking the 1° cell each express secondary (2°) fates in response to lin-12-mediated lateral signaling. The remaining three VPCs each adopt the non- vulval tertiary (3°) fate. Here I describe experiments examining how the selection of these vulval fates is affected by cell cycle arrest and cell cycle-restricted lin-12 activity. The results suggest that lin-12 participates in two

INTRODUCTION

Cell-cell signaling is …

Genetic Control Of Programmed Cell Death In The Caenorhabditis Elegans Hermaphrodite Germline, Tina Gumienny, Eric Lambie, Erika Hartwieg, H. Robert Horvitz, Michael Hengartner

Dartmouth Scholarship

Development of the nematode Caenorhabditis elegans is highly reproducible and the fate of every somatic cell has been reported. We describe here a previously uncharacterized cell fate in C. elegans: we show that germ cells, which in hermaphrodites can differentiate into sperm and oocytes, also undergo apoptotic cell death. In adult hermaphrodites, over 300 germ cells die, using the same apoptotic execution machinery (ced-3, ced-4 and ced-9) as the previously described 131 somatic cell deaths. However, this machinery is activated by a distinct pathway, as loss of egl-1 function, which inhibits somatic cell death, does not affect germ cell apoptosis. …

Drosophila Fascin Mutants Are Rescued By Overexpression Of The Villin-Like Protein, Quail, Kelly Cant, Brenda A. Knowles, Shalina Mahajan-Miklos, Matthew Heintzelman, Lynn Cooley

Dartmouth Scholarship

Actin bundle assembly in specialized structures such as microvilli on intestinal epithelia and Drosophila bristles requires two actin bundling proteins. In these systems, the distinct biochemical properties and temporal localization of actin bundling proteins suggest that these proteins are not redundant. During Drosophila oogenesis, the formation of cytoplasmic actin bundles in nurse cells requires two actin bundling proteins, fascin encoded by the singed gene and a villin-like protein encoded by the quail gene. singed and quail mutations are fully recessive and each mutation disrupts nurse cell cytoplasmic actin bundle formation. We used P-element mediated germline transformation to overexpress quail in …

Transport Of Er Vesicles On Actin Filaments In Neurons By Myosin V, Joel S. Tabb, Bradley J. Molyneaux, Darien L. Cohen, Sergei A. Kuznetsov, George M. Langford

Dartmouth Scholarship

Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was …

Transport Of Axl2p Depends On Erv14p, An Er–Vesicle Protein Related To The Drosophila Cornichon Gene Product, Jacqueline Powers, Charles Barlowe

Dartmouth Scholarship

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER-vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not …

Developmental Regulation Of A Cyclin-Dependent Kinase Inhibitor Controls Postembryonic Cell Cycle Progression In Caenorhabditis Elegans, Yang Hong, Richard Roy, Victor Ambros

Dartmouth Scholarship

C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1 is developmentally regulated in blast cells coincident with G1, and in differentiating cells. Ectopic expression of CKI-1 can prematurely arrest cells in G1, while reducing cki-1 activity by RNA-mediated interference (RNAi) causes extra larval cell divisions, suggesting a role for cki-1 in the developmental control of

INTRODUCTION

The proper development of a multicellular organism requires the precise orchestration of cell proliferation and differentiation. Despite considerable progress toward …

Sec35p, A Novel Peripheral Membrane Protein, Is Required For Er To Golgi Vesicle Docking, Susan M. Vanrheenen, Xiaochun Cao, Vladimir V. Lupashin, Charles Barlowe, M. Gerard Waters

Dartmouth Scholarship

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target …

Coupled Er To Golgi Transport Reconstituted With Purified Cytosolic Proteins, Charles Barlowe

Dartmouth Scholarship

A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-alpha-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi …

A Heterodimer Of Thioredoxin And Ib2 Cooperates With Sec18p (Nsf) To Promote Yeast Vacuole Inheritance, Zuoyu Xu, Andreas Mayer, Eric Muller, William Wickner

Dartmouth Scholarship

Early in S phase, the vacuole (lysosome) of Saccharomyces cerevisiae projects a stream of vesicles and membranous tubules into the bud where they fuse and establish the daughter vacuole. This inheritance reaction can be studied in vitro with isolated vacuoles. Rapid and efficient homotypic fusion between saltwashed vacuoles requires the addition of only two purified soluble proteins, Sec18p (NSF) and LMA1, a novel heterodimer with a thioredoxin subunit. We now report the identity of the second subunit of LMA1 as IB2, a previously identified cytosolic inhibitor of vacuolar proteinase B. Both subunits are needed for efficient vacuole inheritance in vivo …

Numa Assembles Into An Extensive Filamentous Structure When Expressed In The Cell Cytoplasm, Alejandro Saredi, Louisa Howard, Duane A. Compton

Dartmouth Scholarship

NuMA is a 236 kDa protein that participates in the organization of the mitotic spindle despite its strict localization in the nucleus during interphase. To test how cells progress through mitosis when NuMA is localized in the cytoplasm instead of the nucleus, we have deleted the nuclear localization sequence of NuMA using site-directed mutagenesis and transiently expressed this mutant protein (NuMA-DeltaNLS) in BHK-21 cells. During interphase, NuMA-DeltaNLS accumulates in the cytoplasm as a large mass approximately the same size as the cell nucleus. When cells enter mitosis, NuMA-DeltaNLS associates normally with the mitotic spindle without causing any apparent deleterious effects …