Cell Biology Commons^™

A Mitochondria-Anchored Isoform Of The Actin-Nucleating Spire Protein Regulates Mitochondrial Division, Uri Manor, Sadie Bartholomew, Gonen Golani, Eric Christenson, Michael Kozlov, Henry Higgs, James Spudich, Jennifer Lippincott-Schwartz

Dartmouth Scholarship

Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction …

Condensin Ii Promotes The Formation Of Chromosome Territories By Inducing Axial Compaction Of Polyploid Interphase Chromosomes, Christopher R. R. Bauer, Tom A. Hartl, Giovanni Bosco

Dartmouth Scholarship

The eukaryotic nucleus is both spatially and functionally partitioned. This organization contributes to the maintenance, expression, and transmission of genetic information. Though our ability to probe the physical structure of the genome within the nucleus has improved substantially in recent years, relatively little is known about the factors that regulate its organization or the mechanisms through which specific organizational states are achieved. Here, we show that Drosophila melanogaster Condensin II induces axial compaction of interphase chromosomes, globally disrupts interchromosomal interactions, and promotes the dispersal of peri-centric heterochromatin. These Condensin II activities compartmentalize the nucleus into discrete chromosome territories and indicate …

Septin Filaments Exhibit A Dynamic, Paired Organization That Is Conserved From Yeast To Mammals, Bradley S. Demay, Xiaobo Bai, Louisa Howard, Patricia Occhipinti, Rebecca A. Meseroll, Elias T. Spiliotis, Rudolf Oldenbourg, Amy S. Gladfelter

Dartmouth Scholarship

The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy …

Pcdp1 Is A Central Apparatus Protein That Binds Ca2+-Calmodulin And Regulates Ciliary Motility, Christen G. Dipetrillo, Elizabeth F. Smith

Dartmouth Scholarship

For all motile eukaryotic cilia and flagella, beating is regulated by changes in intraciliary calcium concentration. Although the mechanism for calcium regulation is not understood, numerous studies have shown that calmodulin (CaM) is a key axonemal calcium sensor. Using anti-CaM antibodies and Chlamydomonas reinhardtii axonemal extracts, we precipitated a complex that includes four polypeptides and that specifically interacts with CaM in high [Ca²⁺]. One of the complex members, FAP221, is an orthologue of mammalian Pcdp1 (primary ciliary dyskinesia protein 1). Both FAP221 and mammalian Pcdp1 specifically bind CaM in high [Ca²⁺]. Reduced expression of Pcdp1 complex …

Mtorc1 Hyperactivity Inhibits Serum Deprivation-Induced Apoptosis Via Increased Hexokinase Ii And Glut1 Expression, Sustained Mcl-1 Expression, And Glycogen Synthase Kinase 3Β Inhibition, Prashanth T. Bhaskar, Veronique Nogueira, Krushna C. Patra, Sang-Min Jeon, Youngkyu Park, R. Brooks Robey, Nissim Hay

Dartmouth Scholarship

The current concept is that Tsc-deficient cells are sensitized to apoptosis due to the inhibition of Akt activity by the negative feedback mechanism induced by the hyperactive mTORC1. Unexpectedly, however, we found that Tsc1/2-deficient cells exhibit increased resistance to serum deprivation-induced apoptosis. mTORC1 hyperactivity contributes to the apoptotic resistance of serum-deprived Tsc1/2-deficient cells in part by increasing the growth factor-independent expression of hexokinase II (HKII) and GLUT1. mTORC1-mediated increase in hypoxia-inducible factor 1α (HIF1α) abundance, which occurs in the absence of serum in normoxic Tsc2-deficient cells, contributes to these changes. Increased HIF1α abundance in these cells is attributed to both …

A Conserved Cam- And Radial Spoke–Associated Complex Mediates Regulation Of Flagellar Dynein Activity, Erin E. Dymek, Elizabeth F. Smith

Dartmouth Scholarship

For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)- binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which …

Regulation Of Meiotic Cohesion And Chromosome Core Morphogenesis During Pachytene In Drosophila Oocytes, Radhika S. Khetani, Sharon E. Bickel

Dartmouth Scholarship

During meiosis, cohesion between sister chromatids is required for normal levels of homologous recombination, maintenance of chiasmata and accurate chromosome segregation during both divisions. In Drosophila, null mutations in the ord gene abolish meiotic cohesion, although how ORD protein promotes cohesion has remained elusive. We show that SMC subunits of the cohesin complex colocalize with ORD at centromeres of ovarian germ-line cells. In addition, cohesin SMCs and ORD are visible along the length of meiotic chromosomes during pachytene and remain associated with chromosome cores following DNase I digestion. In flies lacking ORD activity, cohesin SMCs fail to accumulate at oocyte …

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 …

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 …

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 …

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 …

Binding Of Matrix Attachment Regions To Lamin Polymers Involves Single-Stranded Regions And The Minor Groove., M. E. Eva Ludérus, Jan L. Den Blaauwen, Oncko J. De Smit, Duane A. Compton, Roel Van Driel

Dartmouth Scholarship

Chromatin in eukaryotic nuclei is thought to be partitioned into functional loop domains that are generated by the binding of defined DNA sequences, named MARs (matrix attachment regions), to the nuclear matrix. We have previously identified B-type lamins as MAR-binding matrix components (M. E. E. Ludérus, A. de Graaf, E. Mattia, J. L. den Blaauwen, M. A. Grande, L. de Jong, and R. van Driel, Cell 70:949-959, 1992). Here we show that A-type lamins and the structurally related proteins desmin and NuMA also specifically bind MARs in vitro. We studied the interaction between MARs and lamin polymers in molecular detail …

Atp-Dependent Formation And Motility Of Aster-Like Structures With Isolated Calf Brain Microtubule Proteins., Richard C. Weisenberg, Robert D. Allen, Shinya Inoue

Dartmouth Scholarship

Microtubule proteins isolated from calf brain will undergo gelation-contraction in the presence of ATP. We have now examined this process by video-enhanced contrast microscopy. After ATP addition to steady-state microtubules, slow (1-5 micron/min), linear movements of particles and microtubules toward aggregation centers occur. The resulting structures resemble mitotic spindle asters. During the time when gel contraction occurs, asters move (at 1-5 micron/min) toward other nearby asters. This is accompanied by the apparent shortening of the microtubules running between the asters. This is the first example of isolated microtubules undergoing a process that has similarities to half-spindle shortening during anaphase A. …

Monoclonal Antibodies To Novel Myeloid Antigens Reveal Human Neutrophil Heterogeneity., Edward D. Ball, Robert F. Graziano, Li Shen, Michael W. Fanger

Dartmouth Scholarship

Three cytotoxic murine monoclonal antibodies that recognize myeloid-specific antigens have been produced by immunization with normal human neutrophils or myeloblasts from a patient with acute myelomonocytic leukemia. Two of these, PMN 6 and PMN 29, are specific for neutrophils; the third monoclonal antibody, AML-2-23, is reactive with the majority of normal monocytes as well as a subpopulation of mature neutrophils. Although neutrophils from all individuals tested expressed these antigens, cytofluorographic analysis revealed that the percentage of cells bearing the PMN 6 and AML-2-23 antigens varied among individuals. Significant additional heterogeneity in the density of each antigen among antigen-bearing cells was …