scholarly journals Cell cycle–regulated membrane binding of NuMA contributes to efficient anaphase chromosome separation

2014 ◽  
Vol 25 (5) ◽  
pp. 606-619 ◽  
Author(s):  
Zhen Zheng ◽  
Qingwen Wan ◽  
Gerry Meixiong ◽  
Quansheng Du
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Membrane Binding ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Α Subunit ◽  
Definitive Evidence ◽  
C Terminus ◽  
Chromosome Separation ◽  
Spindle Elongation

Accurate and efficient separation of sister chromatids during anaphase is critical for faithful cell division. It has been proposed that cortical dynein–generated pulling forces on astral microtubules contribute to anaphase spindle elongation and chromosome separation. In mammalian cells, however, definitive evidence for the involvement of cortical dynein in chromosome separation is missing. It is believed that dynein is recruited and anchored at the cell cortex during mitosis by the α subunit of heterotrimeric G protein (Gα)/mammalian homologue of Drosophila Partner of Inscuteable/nuclear mitotic apparatus (NuMA) ternary complex. Here we uncover a Gα/LGN-independent lipid- and membrane-binding domain at the C-terminus of NuMA. We show that the membrane binding of NuMA is cell cycle regulated—it is inhibited during prophase and metaphase by cyclin-dependent kinase 1 (CDK1)–mediated phosphorylation and only occurs after anaphase onset when CDK1 activity is down-regulated. Further studies indicate that cell cycle–regulated membrane association of NuMA underlies anaphase-specific enhancement of cortical NuMA and dynein. By replacing endogenous NuMA with membrane-binding-deficient NuMA, we can specifically reduce the cortical accumulation of NuMA and dynein during anaphase and demonstrate that cortical NuMA and dynein contribute to efficient chromosome separation in mammalian cells.

scholarly journals Evidence for dynein and astral microtubule–mediated cortical release and transport of Gαi/LGN/NuMA complex in mitotic cells

2013 ◽  
Vol 24 (7) ◽  
pp. 901-913 ◽  
Author(s):  
Zhen Zheng ◽  
Qingwen Wan ◽  
Jing Liu ◽  
Huabin Zhu ◽  
Xiaogang Chu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Fluorescence Recovery After Photobleaching ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Spindle Positioning ◽  
Spindle Poles ◽  
Astral Microtubule ◽  
Mammalian Homologue ◽  
Cortical Localization

Spindle positioning is believed to be governed by the interaction between astral microtubules and the cell cortex and involve cortically anchored motor protein dynein. How dynein is recruited to and regulated at the cell cortex to generate forces on astral microtubules is not clear. Here we show that mammalian homologue of Drosophila Pins (Partner of Inscuteable) (LGN), a Gαi-binding protein that is critical for spindle positioning in different systems, associates with cytoplasmic dynein heavy chain (DYNC1H1) in a Gαi-regulated manner. LGN is required for the mitotic cortical localization of DYNC1H1, which, in turn, also modulates the cortical accumulation of LGN. Using fluorescence recovery after photobleaching analysis, we show that cortical LGN is dynamic and the turnover of LGN relies, at least partially, on astral microtubules and DYNC1H1. We provide evidence for dynein- and astral microtubule–mediated transport of Gαi/LGN/nuclear mitotic apparatus (NuMA) complex from cell cortex to spindle poles and show that actin filaments counteract such transport by maintaining Gαi/LGN/NuMA and dynein at the cell cortex. Our results indicate that astral microtubules are required for establishing bipolar, symmetrical cortical LGN distribution during metaphase. We propose that regulated cortical release and transport of LGN complex along astral microtubules may contribute to spindle positioning in mammalian cells.

scholarly journals SLK-dependent activation of ERMs controls LGN–NuMA localization and spindle orientation

2014 ◽  
Vol 205 (6) ◽  
pp. 791-799 ◽  
Author(s):  
Mickael Machicoane ◽  
Cristina A. de Frutos ◽  
Jenny Fink ◽  
Murielle Rocancourt ◽  
Yannis Lombardi ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Molecular Level ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Mitotic Entry ◽  
Spindle Microtubules ◽  
Force Generator

Mitotic spindle orientation relies on a complex dialog between the spindle microtubules and the cell cortex, in which F-actin has been recently implicated. Here, we report that the membrane–actin linkers ezrin/radixin/moesin (ERMs) are strongly and directly activated by the Ste20-like kinase at mitotic entry in mammalian cells. Using microfabricated adhesive substrates to control the axis of cell division, we found that the activation of ERMs plays a key role in guiding the orientation of the mitotic spindle. Accordingly, impairing ERM activation in apical progenitors of the mouse embryonic neocortex severely disturbed spindle orientation in vivo. At the molecular level, ERM activation promotes the polarized association at the mitotic cortex of leucine-glycine-asparagine repeat protein (LGN) and nuclear mitotic apparatus (NuMA) protein, two essential factors for spindle orientation. We propose that activated ERMs, together with Gαi, are critical for the correct localization of LGN–NuMA force generator complexes and hence for proper spindle orientation.

scholarly journals Cell cycle–regulated cortical dynein/dynactin promotes symmetric cell division by differential pole motion in anaphase

2012 ◽  
Vol 23 (17) ◽  
pp. 3380-3390 ◽  
Author(s):  
Elizabeth S. Collins ◽  
Sai Keshavan Balchand ◽  
Jessica L. Faraci ◽  
Patricia Wadsworth ◽  
Wei-Lih Lee
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Affinity Purification ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Spindle Positioning ◽  
Artificial Chromosomes ◽  
Pole Motion ◽  
Mammalian Cell Lines ◽  
In Cells

In cultured mammalian cells, how dynein/dynactin contributes to spindle positioning is poorly understood. To assess the role of cortical dynein/dynactin in this process, we generated mammalian cell lines expressing localization and affinity purification (LAP)–tagged dynein/dynactin subunits from bacterial artificial chromosomes and observed asymmetric cortical localization of dynein and dynactin during mitosis. In cells with asymmetrically positioned spindles, dynein and dynactin were both enriched at the cortex distal to the spindle. NuMA, an upstream targeting factor, localized asymmetrically along the cell cortex in a manner similar to dynein and dynactin. During spindle motion toward the distal cortex, dynein and dynactin were locally diminished and subsequently enriched at the new distal cortex. At anaphase onset, we observed a transient increase in cortical dynein, followed by a reduction in telophase. Spindle motion frequently resulted in cells entering anaphase with an asymmetrically positioned spindle. These cells gave rise to symmetric daughter cells by dynein-dependent differential spindle pole motion in anaphase. Our results demonstrate that cortical dynein and dynactin dynamically associate with the cell cortex in a cell cycle–regulated manner and are required to correct spindle mispositioning in LLC-Pk1 epithelial cells.

scholarly journals The role of isoprenylation in membrane attachment of nuclear lamins. A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties

1994 ◽  
Vol 107 (4) ◽  
pp. 1019-1029 ◽  
Author(s):  
H. Hennekes ◽  
E.A. Nigg
Keyword(s):  
Mammalian Cells ◽  
Membrane Binding ◽  
Proteolytic Cleavage ◽  
Single Point Mutation ◽  
Lamin A ◽  
Binding Properties ◽  
Nuclear Lamins ◽  
C Terminus ◽  
Membrane Attachment

Mature A- and B-type lamins differ in the extent to which they interact with the nuclear membrane and thus represent an interesting model for studying the role of isoprenylation and carboxyl-methylation in membrane attachment. Both A- and B-type lamins are isoprenylated and carboxyl-methylated shortly after synthesis, but A-type lamins undergo a further proteolytic cleavage which results in the loss of the hydrophobically modified C terminus. Here, we have constructed mutants of chicken lamin A that differ in their abilities to serve as substrates for different post-translational processing events occurring at the C terminus of the wild-type precursor. In addition to studying full-length proteins, we have analyzed C-terminal end domains of lamin A, either alone or after fusion to reporter proteins. Mutant proteins were expressed in mammalian cells, and their membrane association was analyzed by immunofluorescence microscopy and subcellular fractionation. Our results provide information on the substrate specificity and subcellular localization of the lamin-A-specific protease. Moreover, they indicate that hydrophobic modifications of the C-terminal end domains account for the differential membrane-binding properties of A- and B-type lamins. Thus, some of the integral membrane proteins implicated in anchoring B-type lamins to the membrane may function as receptors for the isoprenylated and carboxyl-methylated C terminus.

Regulation of Microtubule Assembly-Disassembly in Mitotic Mammalian Cells: Role of Calcium, Calmodulin and MTOCs

1981 ◽  
Vol 39 ◽  
pp. 572-575
Author(s):  
B. R. Brinkley ◽  
S. L. Brenner ◽  
D. A. Pepper ◽  
R. L. Pardue
Keyword(s):  
Cell Cycle ◽  
Calcium Binding ◽  
Mammalian Cells ◽  
Microtubule Assembly ◽  
Free Calcium ◽  
Mitotic Apparatus ◽  
Cytoplasmic Microtubule ◽  
Dividing Cells ◽  
Late Telophase

Two microtubule arrays exist in cultured mammalian cells during their progression through the cell cycle; the cytoplasmic microtubule complexes (CMTC) of interphase cells (Figure 1) and the mitotic apparatus (MA) of dividing cells (Figure 2). As chromosomes are segregated to opposite poles of the spindle during telophase, the microtubules of the MA are disassembled. During late telophase -G1 phase the tubulin subunits from the spindle are recycled into the microtubules of the CMTC which forms an elaborate network throught the cytoplasm. When cells progress into late G2 -Prophase, the CMTC is disassembled and the tubulin is converted into microtubules of the MA. our research has been aimed at defining the mechanism whereby cells regulate the alternating patterns of microtubule assembly-disassembly during the cell cycle.In one series of experiments, we have investigated the role of calcium in microtubule assembly. Several laboratories have shown that cytoplasmic and spindle microtubules are unstable in the presence of elevated free calcium levels. Using monospecific antibodies and indirect immunofluorescence, we have demonstrated the presence of the ubiquitous calcium-binding protein calmodulin in the mitotic spindle of mammalian cells in vitro (Figure 3).

Spindle scaling mechanisms

2020 ◽  
Vol 64 (2) ◽  
pp. 383-396
Author(s):  
Lara K. Krüger ◽  
Phong T. Tran
Keyword(s):  
Cell Size ◽  
Spindle Assembly ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Size Dependent ◽  
A Cell ◽  
Chromosome Separation ◽  
Spindle Elongation ◽  
Protein Gradients ◽  
Spindle Structure

Abstract The mitotic spindle robustly scales with cell size in a plethora of different organisms. During development and throughout evolution, the spindle adjusts to cell size in metazoans and yeast in order to ensure faithful chromosome separation. Spindle adjustment to cell size occurs by the scaling of spindle length, spindle shape and the velocity of spindle assembly and elongation. Different mechanisms, depending on spindle structure and organism, account for these scaling relationships. The limited availability of critical spindle components, protein gradients, sequestration of spindle components, or post-translational modification and differential expression levels have been implicated in the regulation of spindle length and the spindle assembly/elongation velocity in a cell size-dependent manner. In this review, we will discuss the phenomenon and mechanisms of spindle length, spindle shape and spindle elongation velocity scaling with cell size.

Centrosome Structure And Function Is Altered By Experimental Manipulations With Formamide: Implications For Abnormal Cell Divisions During Cancer

1999 ◽  
Vol 5 (S2) ◽  
pp. 1286-1287
Author(s):  
Heide Schatten ◽  
Christopher N. Hueser ◽  
Amitabha Chakrabarti
Keyword(s):  
Cell Cycle ◽  
Genomic Stability ◽  
Cellular Regulation ◽  
Mitotic Apparatus ◽  
Abnormal Cell ◽  
Chemical Agents ◽  
Abnormal Mitosis ◽  
And Function ◽  
Normal Mitosis ◽  
Abnormal Cell Proliferation

The formation of abnormal mitosis associated with cancer has been intriguing for many decades. While microtubules had been the focus of previous studies, recent research has focused on centrosomes, microtubule organizing centers which organize the mitotic apparatus during cell division. During normal mitosis centrosomes form two poles but in cancer, centrosomes can form three, four, or more poles, and organize tripolar, quadripolar, and multipolar mitoses, respectively. This has severe consequences for genomic stability because chromosomes are separated unequally to three, four, or more poles. This can result in aneuploidy and gene amplifications with multiple defects in cellular regulation. It can result in malignancy that is accompanied by cell cycle imbalances and abnormal cell proliferation. While radiation and chemical agents are known to damage DNA and can lead to cell cycle abnormalities, the damage of centrosome structure leading to abnormal mitosis deserves also consideration.

scholarly journals Phosphorylation of the RB C-terminus regulates condensin II release from chromatin

2020 ◽  
pp. jbc.RA120.016511
Author(s):  
Seung J Kim ◽  
James I MacDonald ◽  
Frederick A. Dick
Keyword(s):  
Cell Cycle ◽  
Genome Stability ◽  
Cell Cycle Control ◽  
Cell Receptor ◽  
Receptor Activation ◽  
Biologically Relevant ◽  
C Terminus ◽  
Independent Functions ◽  
Phosphorylation Event ◽  
Rb Phosphorylation

The retinoblastoma tumour suppressor protein (RB) plays an important role in biological processes such as cell cycle control, DNA damage repair, epigenetic regulation, and genome stability. The canonical model of RB regulation is that cyclin-CDKs phosphorylate, and render RB inactive in late G1/S, promoting entry into S phase. Recently, mono-phosphorylated RB species were described to have distinct cell-cycle independent functions, suggesting that a phosphorylation code dictates diversity of RB function. However, a biologically relevant, functional role of RB phosphorylation at non-CDK sites has remained elusive. Here, we investigated S838/T841 dual phosphorylation, its upstream stimulus, and downstream functional output.  We found that mimicking T-cell receptor activation in Jurkat leukemia cells induced sequential activation of downstream kinases including p38 MAPK, and RB S838/T841 phosphorylation.  This signaling pathway disrupts RB and condensin II interaction with chromatin.  Using cells expressing a WT or S838A/T841A mutant RB fragment, we present evidence that deficiency for this phosphorylation event prevents condensin II release from chromatin.

scholarly journals A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3317
Author(s):  
Eric Moeglin ◽  
Dominique Desplancq ◽  
Audrey Stoessel ◽  
Christian Massute ◽  
Jeremy Ranniger ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Chromatin Modification ◽  
Replication Stress ◽  
Living Cells ◽  
Single Step ◽  
Dna Double Strand Breaks ◽  
Drug Induced ◽  
Histone H2ax ◽  
C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

Sign in / Sign up

Export Citation Format

Share Document