scholarly journals A novel tropomyosin isoform functions at the mitotic spindle and Golgi in Drosophila

2015 ◽  
Vol 26 (13) ◽  
pp. 2491-2504 ◽  
Author(s):  
Lauren M. Goins ◽  
R. Dyche Mullins
Keyword(s):  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Molecular Genetic ◽  
Actin Binding ◽  
Cell Cortex ◽  
Developmental Defects ◽  
C Terminus ◽  
Sequence Elements ◽  
Dividing Cells ◽  
Cytoskeletal Networks

Most eukaryotic cells express multiple isoforms of the actin-binding protein tropomyosin that help construct a variety of cytoskeletal networks. Only one nonmuscle tropomyosin (Tm1A) has previously been described in Drosophila, but developmental defects caused by insertion of P-elements near tropomyosin genes imply the existence of additional, nonmuscle isoforms. Using biochemical and molecular genetic approaches, we identified three tropomyosins expressed in Drosophila S2 cells: Tm1A, Tm1J, and Tm2A. The Tm1A isoform localizes to the cell cortex, lamellar actin networks, and the cleavage furrow of dividing cells—always together with myosin-II. Isoforms Tm1J and Tm2A colocalize around the Golgi apparatus with the formin-family protein Diaphanous, and loss of either isoform perturbs cell cycle progression. During mitosis, Tm1J localizes to the mitotic spindle, where it promotes chromosome segregation. Using chimeras, we identified the determinants of tropomyosin localization near the C-terminus. This work 1) identifies and characterizes previously unknown nonmuscle tropomyosins in Drosophila, 2) reveals a function for tropomyosin in the mitotic spindle, and 3) uncovers sequence elements that specify isoform-specific localizations and functions of tropomyosin.

scholarly journals LGN regulates mitotic spindle orientation during epithelial morphogenesis

2010 ◽  
Vol 189 (2) ◽  
pp. 275-288 ◽  
Author(s):  
Zhen Zheng ◽  
Huabin Zhu ◽  
Qingwen Wan ◽  
Jing Liu ◽  
Zhuoni Xiao ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Mdck Cells ◽  
Apical Cell ◽  
Cell Polarization ◽  
Epithelial Morphogenesis ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Dividing Cells ◽  
Cortical Localization ◽  
Lateral Cell

Coordinated cell polarization and mitotic spindle orientation are thought to be important for epithelial morphogenesis. Whether spindle orientation is indeed linked to epithelial morphogenesis and how it is controlled at the molecular level is still unknown. Here, we show that the NuMA- and Gα-binding protein LGN is required for directing spindle orientation during cystogenesis of MDCK cells. LGN localizes to the lateral cell cortex, and is excluded from the apical cell cortex of dividing cells. Depleting LGN, preventing its cortical localization, or disrupting its interaction with endogenous NuMA or Gα proteins all lead to spindle misorientation and abnormal cystogenesis. Moreover, artificial mistargeting of endogenous LGN to the apical membrane results in a near 90° rotation of the spindle axis and profound cystogenesis defects that are dependent on cell division. The normal apical exclusion of LGN during mitosis appears to be mediated by atypical PKC. Thus, cell polarization–mediated spatial restriction of spindle orientation determinants is critical for epithelial morphogenesis.

scholarly journals The nucleolar RNA methyltransferase Misu (NSun2) is required for mitotic spindle stability

2009 ◽  
Vol 186 (1) ◽  
pp. 27-40 ◽  
Author(s):  
Shobbir Hussain ◽  
Sandra Blanco Benavente ◽  
Elisabete Nascimento ◽  
Ilaria Dragoni ◽  
Agata Kurowski ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Multipolar Spindles ◽  
Direct Target Gene ◽  
Dividing Cells ◽  
Direct Target ◽  
Spindle Stability ◽  
Sun Domain ◽  
Nucleolar Rna

Myc-induced SUN domain–containing protein (Misu or NSun2) is a nucleolar RNA methyltransferase important for c-Myc–induced proliferation in skin, but the mechanisms by which Misu contributes to cell cycle progression are unknown. In this study, we demonstrate that Misu translocates from the nucleoli in interphase to the spindle in mitosis as an RNA–protein complex that includes 18S ribosomal RNA. Functionally, depletion of Misu caused multiple mitotic defects, including formation of unstructured spindles, multipolar spindles, and chromosome missegregation, leading to aneuploidy and cell death. The presence of both RNA and Misu is required for correct spindle assembly, and this process is independent of active translation. Misu might mediate its function at the spindle by recruiting nucleolar and spindle-associated protein (NuSAP), an essential microtubule-stabilizing and bundling protein. We further identify NuSAP as a novel direct target gene of c-Myc. Collectively, our results suggest a novel mechanism by which c-Myc promotes proliferation by stabilizing the mitotic spindle in fast-dividing cells via Misu and NuSAP.

scholarly journals Diverse mitotic functions of the cytoskeletal cross-linking protein Shortstop suggest a role in Dynein/Dynactin activity

2017 ◽  
Vol 28 (19) ◽  
pp. 2555-2568 ◽  
Author(s):  
Evan B. Dewey ◽  
Christopher A. Johnston
Keyword(s):  
Cell Fate ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Epithelial Mesenchymal Transition ◽  
Spindle Pole ◽  
Actin Binding ◽  
Epithelial Tissue ◽  
Mesenchymal Transition ◽  
Underlying Mechanisms ◽  
Dynactin Complex

Proper assembly and orientation of the bipolar mitotic spindle is critical to the fidelity of cell division. Mitotic precision fundamentally contributes to cell fate specification, tissue development and homeostasis, and chromosome distribution within daughter cells. Defects in these events are thought to contribute to several human diseases. The underlying mechanisms that function in spindle morphogenesis and positioning remain incompletely defined, however. Here we describe diverse roles for the actin-microtubule cross-linker Shortstop (Shot) in mitotic spindle function in Drosophila. Shot localizes to mitotic spindle poles, and its knockdown results in an unfocused spindle pole morphology and a disruption of proper spindle orientation. Loss of Shot also leads to chromosome congression defects, cell cycle progression delay, and defective chromosome segregation during anaphase. These mitotic errors trigger apoptosis in Drosophila epithelial tissue, and blocking this apoptotic response results in a marked induction of the epithelial–mesenchymal transition marker MMP-1. The actin-binding domain of Shot directly interacts with Actin-related protein-1 (Arp-1), a key component of the Dynein/Dynactin complex. Knockdown of Arp-1 phenocopies Shot loss universally, whereas chemical disruption of F-actin does so selectively. Our work highlights novel roles for Shot in mitosis and suggests a mechanism involving Dynein/Dynactin activation.

scholarly journals Specific polar subpopulations of astral microtubules control spindle orientation and symmetric neural stem cell division

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Felipe Mora-Bermúdez ◽  
Fumio Matsuzaki ◽  
Wieland B Huttner
Keyword(s):  
Cell Division ◽  
Basal Cell ◽  
Mitotic Spindle ◽  
Asymmetric Cell Division ◽  
Spindle Cell ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Dividing Cells ◽  
Stem Cell Division

Mitotic spindle orientation is crucial for symmetric vs asymmetric cell division and depends on astral microtubules. Here, we show that distinct subpopulations of astral microtubules exist, which have differential functions in regulating spindle orientation and division symmetry. Specifically, in polarized stem cells of developing mouse neocortex, astral microtubules reaching the apical and basal cell cortex, but not those reaching the central cell cortex, are more abundant in symmetrically than asymmetrically dividing cells and reduce spindle orientation variability. This promotes symmetric divisions by maintaining an apico-basal cleavage plane. The greater abundance of apical/basal astrals depends on a higher concentration, at the basal cell cortex, of LGN, a known spindle-cell cortex linker. Furthermore, newly developed specific microtubule perturbations that selectively decrease apical/basal astrals recapitulate the symmetric-to-asymmetric division switch and suffice to increase neurogenesis in vivo. Thus, our study identifies a novel link between cell polarity, astral microtubules, and spindle orientation in morphogenesis.

scholarly journals The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition

2013 ◽  
Vol 24 (18) ◽  
pp. 2885-2893 ◽  
Author(s):  
Derek A. Applewhite ◽  
Kyle D. Grode ◽  
Mara C. Duncan ◽  
Stephen L. Rogers
Keyword(s):  
Actin Binding ◽  
Cell Cortex ◽  
Cross Linking ◽  
Inhibitory Mechanism ◽  
Cross Link ◽  
Inactive Conformation ◽  
Closed Conformation ◽  
Actin Binding Domain ◽  
Ef Hand ◽  
Cytoskeletal Networks

Actin and microtubule dynamics must be precisely coordinated during cell migration, mitosis, and morphogenesis—much of this coordination is mediated by proteins that physically bridge the two cytoskeletal networks. We have investigated the regulation of the Drosophila actin-microtubule cross-linker Short stop (Shot), a member of the spectraplakin family. Our data suggest that Shot's cytoskeletal cross-linking activity is regulated by an intramolecular inhibitory mechanism. In its inactive conformation, Shot adopts a “closed” conformation through interactions between its NH2-terminal actin-binding domain and COOH-terminal EF-hand-GAS2 domain. This inactive conformation is targeted to the growing microtubule plus end by EB1. On activation, Shot binds along the microtubule through its COOH-terminal GAS2 domain and binds to actin with its NH2-terminal tandem CH domains. We propose that this mechanism allows Shot to rapidly cross-link dynamic microtubules in response to localized activating signals at the cell cortex.

Osmotic stress-induced remodeling of the cortical cytoskeleton

2002 ◽  
Vol 283 (3) ◽  
pp. C850-C865 ◽  
Author(s):  
Caterina Di Ciano ◽  
Zilin Nie ◽  
Katalin Szászi ◽  
Alison Lewis ◽  
Takehito Uruno ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
De Novo ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Actin Binding ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Signaling Mechanism ◽  
Actin Binding Domain ◽  
Cytoskeleton Remodeling

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and 3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.

scholarly journals Lis1 is essential for cortical microtubule organization and desmosome stability in the epidermis

2011 ◽  
Vol 194 (4) ◽  
pp. 631-642 ◽  
Author(s):  
Kaelyn D. Sumigray ◽  
Hsin Chen ◽  
Terry Lechler
Keyword(s):  
Cortical Microtubule ◽  
Cell Types ◽  
Epidermal Differentiation ◽  
Cell Cortex ◽  
Microtubule Organization ◽  
Specific Subset ◽  
Keratin Filaments ◽  
Perinatal Lethality ◽  
Cytoskeletal Networks ◽  
Centrosomal Proteins

Desmosomes are cell–cell adhesion structures that integrate cytoskeletal networks. In addition to binding intermediate filaments, the desmosomal protein desmoplakin (DP) regulates microtubule reorganization in the epidermis. In this paper, we identify a specific subset of centrosomal proteins that are recruited to the cell cortex by DP upon epidermal differentiation. These include Lis1 and Ndel1, which are centrosomal proteins that regulate microtubule organization and anchoring in other cell types. This recruitment was mediated by a region of DP specific to a single isoform, DPI. Furthermore, we demonstrate that the epidermal-specific loss of Lis1 results in dramatic defects in microtubule reorganization. Lis1 ablation also causes desmosomal defects, characterized by decreased levels of desmosomal components, decreased attachment of keratin filaments, and increased turnover of desmosomal proteins at the cell cortex. This contributes to loss of epidermal barrier activity, resulting in completely penetrant perinatal lethality. This work reveals essential desmosome-associated components that control cortical microtubule organization and unexpected roles for centrosomal proteins in epidermal function.

scholarly journals Villidin, a Novel WD-repeat and Villin-related Protein fromDictyostelium,Is Associated with Membranes and the Cytoskeleton

2003 ◽  
Vol 14 (7) ◽  
pp. 2716-2727 ◽  
Author(s):  
Annika Gloss ◽  
Francisco Rivero ◽  
Nandkumar Khaire ◽  
Rolf Müller ◽  
William F. Loomis ◽  
...  
Keyword(s):  
Actin Binding ◽  
Similar Distribution ◽  
Fruiting Bodies ◽  
Targeted Deletion ◽  
C Terminus ◽  
Its Gene ◽  
Mutant Strains ◽  
Protein Functions ◽  
Wd Repeats ◽  
Actin Binding Site

Villidin is a novel multidomain protein (190 kDa) from Dictyostelium amoebae containing WD repeats at its N-terminus, three PH domains in the middle of the molecule, and five gelsolin-like segments at the C-terminus, followed by a villin-like headpiece. Villidin mRNA and protein are present in low amounts during growth and early aggregation, but increase during development and reach their highest levels at the tipped mound stage. The protein is present in the cytosol as well as in the cytoskeletal and membrane fractions. GFP-tagged full-length villidin exhibits a similar distribution as native villidin, including a distinct colocalization with Golgi structures. Interestingly, GFP fusions with the gelsolin/villin-like region are uniformly dispersed in the cytoplasm, whereas GFP fusions of the N-terminal WD repeats codistribute with F-actin and are associated with the Triton-insoluble cytoskeleton. Strains lacking villidin because of targeted deletion of its gene grow normally and can develop into fruiting bodies. However, cell motility is reduced during aggregation and phototaxis is impaired in the mutant strains. We conclude that villidin harbors a major F-actin binding site in the N-terminal domain and not in the villin-like region as expected; association of villidin with vesicular membranes suggests that the protein functions as a linker between membranes and the actin cytoskeleton.

scholarly journals The Paramyxovirus Simian Virus 5 V Protein Slows Progression of the Cell Cycle

2000 ◽  
Vol 74 (19) ◽  
pp. 9152-9166 ◽  
Author(s):  
Grace Y. Lin ◽  
Robert A. Lamb
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Simian Virus ◽  
S Phase ◽  
V Protein ◽  
Simian Virus 5 ◽  
C Terminus ◽  
M Phase ◽  
Infected Cells ◽  
Affect Cell Cycle Progression

ABSTRACT Infection of cells by many viruses affects the cell division cycle of the host cell to favor viral replication. We examined the ability of the paramyxovirus simian parainfluenza virus 5 (SV5) to affect cell cycle progression, and we found that SV5 slows the rate of proliferation of HeLa T4 cells. The SV5-infected cells had a delayed transition from G1 to S phase and prolonged progression through S phase, and some of the infected cells were arrested in G2 or M phase. The levels of p53 and p21CIP1were not increased in SV5-infected cells compared to mock-infected cells, suggesting that the changes in the cell cycle occur through a p53-independent mechanism. However, the phosphorylation of the retinoblastoma protein (pRB) was delayed and prolonged in SV5-infected cells. The changes in the cell cycle were also observed in cells expressing the SV5 V protein but not in the cells expressing the SV5 P protein or the V protein lacking its unique C terminus (VΔC). The unique C terminus of the V protein of SV5 was shown previously to interact with DDB1, which is the 127-kDa subunit of the multifunctional damage-specific DNA-binding protein (DDB) heterodimer. The coexpression of DDB1 with V can partially restore the changes in the cell cycle caused by expression of the V protein.

scholarly journals Dosage-Dependent Effects of Akt1/Protein Kinase Bα (PKBα) and Akt3/PKBγ on Thymus, Skin, and Cardiovascular and Nervous System Development in Mice

2005 ◽  
Vol 25 (23) ◽  
pp. 10407-10418 ◽  
Author(s):  
Zhong-Zhou Yang ◽  
Oliver Tschopp ◽  
Nicolas Di-Poï ◽  
Elisabeth Bruder ◽  
Anne Baudry ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cell Cycle Progression ◽  
System Development ◽  
Critical Role ◽  
Nervous System Development ◽  
Mouse Development ◽  
Double Knockout ◽  
Developmental Defects ◽  
Regulation Of Metabolism ◽  
Survival And Development

ABSTRACT Akt/protein kinase B (PKB) plays a critical role in the regulation of metabolism, transcription, cell migration, cell cycle progression, and cell survival. The existence of viable knockout mice for each of the three isoforms suggests functional redundancy. We generated mice with combined mutant alleles of Akt1 and Akt3 to study their effects on mouse development. Here we show that Akt1 − / − Akt3 +/ − mice display multiple defects in the thymus, heart, and skin and die within several days after birth, while Akt1 +/ − Akt3 − / − mice survive normally. Double knockout (Akt1 − / − Akt3 − / −) causes embryonic lethality at around embryonic days 11 and 12, with more severe developmental defects in the cardiovascular and nervous systems. Increased apoptosis was found in the developing brain of double mutant embryos. These data indicate that the Akt1 gene is more essential than Akt3 for embryonic development and survival but that both are required for embryo development. Our results indicate isoform-specific and dosage-dependent effects of Akt on animal survival and development.

Sign in / Sign up

Export Citation Format

Share Document