Inscuteable-dependent apical localization of the microtubule-binding protein Cornetto suggests a role in asymmetric cell division

2001 ◽  
Vol 114 (20) ◽  
pp. 3655-3662 ◽  
Author(s):  
Silvia Bulgheresi ◽  
Elke Kleiner ◽  
Juergen A. Knoblich
Keyword(s):  
Cell Fate ◽  
Mitotic Spindle ◽  
Binding Protein ◽  
Protein Localization ◽  
Apical Cell ◽  
Coiled Coil ◽  
Cell Cortex ◽  
Dependent Manner ◽  
Genetic Redundancy ◽  
Microtubule Binding

Drosophila neuroblasts divide asymmetrically along the apical-basal axis. The Inscuteable protein localizes to the apical cell cortex in neuroblasts from interphase to metaphase, but disappears in anaphase. Inscuteable is required for correct spindle orientation and for asymmetric localization of cell fate determinants to the opposite (basal) cell cortex. Here, we show that Inscuteable also directs asymmetric protein localization to the apical cell cortex during later stages of mitosis. In a two-hybrid screen for Inscuteable-binding proteins, we have identified the coiled-coil protein Cornetto, which shows a highly unusual subcellular distribution in neuroblasts. Although the protein is uniformly distributed in the cytoplasm during metaphase, it concentrates apically in anaphase and forms an apical crescent during telophase in an inscuteable-dependent manner. Upon overexpression, Cornetto localizes to astral microtubules and microtubule spin-down experiments demonstrate that Cornetto is a microtubule-binding protein. After disruption of the actin cytoskeleton, Cornetto localizes with microtubules throughout the cell cycle and decorates the mitotic spindle during metaphase. Our results reveal a novel pattern of asymmetric protein localization in Drosophila neuroblasts and are consistent with a function of Cornetto in anchoring the mitotic spindle during late phases of mitosis, even though our cornetto mutant analysis suggests that this function might be obscured by genetic redundancy.

A functional analysis of inscuteable and its roles during Drosophila asymmetric cell divisions

1999 ◽  
Vol 112 (10) ◽  
pp. 1541-1551 ◽  
Author(s):  
M. Tio ◽  
M. Zavortink ◽  
X. Yang ◽  
W. Chia
Keyword(s):  
Cell Fate ◽  
Mitotic Spindle ◽  
Mother Cell ◽  
Apical Cell ◽  
Cell Cortex ◽  
Asymmetric Cell Divisions ◽  
Cell Divisions ◽  
Asymmetric Divisions ◽  
Necessary And Sufficient ◽  
Ganglion Mother Cell

Cellular diversity in the Drosophila central nervous system is generated through a series of asymmetric cell divisions in which one progenitor produces two daughter cells with distinct fates. Asymmetric basal cortical localisation and segregation of the determinant Prospero during neuroblast cell divisions play a crucial role in effecting distinct cell fates for the progeny sibling neuroblast and ganglion mother cell. Similarly asymmetric localisation and segregation of the determinant Numb during ganglion mother cell divisions ensure that the progeny sibling neurons attain distinct fates. The most upstream component identified so far which acts to organise both neuroblast and ganglion mother cell asymmetric divisions is encoded by inscuteable. The Inscuteable protein is itself asymmetrically localised to the apical cell cortex and is required both for the basal localisation of the cell fate determinants during mitosis and for the orientation of the mitotic spindle along the apical/basal axis. Here we define the functional domains of Inscuteable. We show that aa252-578 appear sufficient to effect all aspects of its function, however, the precise requirements for its various functions differ. The region, aa288-497, is necessary and sufficient for apical cortical localisation and for mitotic spindle (re)orientation along the apical/basal axis. A larger region aa288-540 is necessary and sufficient for asymmetric Numb localisation and segregation; however, correct localisation of Miranda and Prospero requires additional sequences from aa540-578. The requirement for the resolution of distinct sibling neuronal fates appears to coincide with the region necessary and sufficient for Numb localisation (aa288-540). Our data suggest that apical localisation of the Inscuteable protein is a necessary prerequisite for all other aspects of its function. Finally, we show that although inscuteable RNA is normally apically localised, RNA localisation is not required for protein localisation or any aspects of inscuteable function.

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 Bim1p/Yeb1p Mediates the Kar9p-dependent Cortical Attachment of Cytoplasmic Microtubules

2000 ◽  
Vol 11 (9) ◽  
pp. 2949-2959 ◽  
Author(s):  
Rita K. Miller ◽  
Soo-Chen Cheng ◽  
Mark D. Rose
Keyword(s):  
Fluorescent Protein ◽  
Binding Protein ◽  
Spindle Pole Body ◽  
Attachment Site ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Microtubule Binding ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Two Hybrid

In Saccharomyces cerevisiae, positioning of the mitotic spindle depends on the interaction of cytoplasmic microtubules with the cell cortex. In this process, cortical Kar9p in the bud acts as a link between the actin and microtubule cytoskeletons. To identify Kar9p-interacting proteins, a two-hybrid screen was conducted with the use of full-length Kar9p as bait, and three genes were identified: BIM1, STU2, andKAR9 itself. STU2 encodes a component of the spindle pole body. Bim1p is the yeast homologue of the human microtubule-binding protein EB1, which is a binding partner to the adenomatous polyposis coli protein involved in colon cancer. Eighty-nine amino acids within the third quarter of Bim1p was sufficient to confer interaction with Kar9p. The two-hybrid interactions were confirmed with the use of coimmunoprecipitation experiments. Genetic analysis placed Bim1p in the Kar9p pathway for nuclear migration. Bim1p was not required for Kar9p's cortical or spindle pole body localization. However, deletion ofBIM1 eliminated Kar9p localization along cytoplasmic microtubules. Furthermore, in the bim1 mutants, the cytoplasmic microtubules no longer intersected the cortical dot of Green Fluorescent Protein–Kar9p. These experiments demonstrate that the interaction of cytoplasmic microtubules with the Kar9p cortical attachment site requires the microtubule-binding protein Bim1p.

scholarly journals The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

2015 ◽  
Vol 211 (2) ◽  
pp. 309-322 ◽  
Author(s):  
Lindsay G. Lammers ◽  
Steven M. Markus
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Budding Yeast ◽  
Coiled Coil ◽  
Binding Domain ◽  
Cell Cortex ◽  
Wild Type ◽  
Microtubule Binding ◽  
Coiled Coil Domain ◽  
Microtubule Binding Domain

Cortically anchored dynein orients the spindle through interactions with astral microtubules. In budding yeast, dynein is offloaded to Num1 receptors from microtubule plus ends. Rather than walking toward minus ends, dynein remains associated with plus ends due in part to its association with Pac1/LIS1, an inhibitor of dynein motility. The mechanism by which dynein is switched from “off” at the plus ends to “on” at the cell cortex remains unknown. Here, we show that overexpression of the coiled-coil domain of Num1 specifically depletes dynein–dynactin–Pac1/LIS1 complexes from microtubule plus ends and reduces dynein-Pac1/LIS1 colocalization. Depletion of dynein from plus ends requires its microtubule-binding domain, suggesting that motility is required. An enhanced Pac1/LIS1 affinity mutant of dynein or overexpression of Pac1/LIS1 rescues dynein plus end depletion. Live-cell imaging reveals minus end–directed dynein–dynactin motility along microtubules upon overexpression of the coiled-coil domain of Num1, an event that is not observed in wild-type cells. Our findings indicate that dynein activity is directly switched “on” by Num1, which induces Pac1/LIS1 removal.

scholarly journals Polarization and cell-fate decision facilitated by the adaptor Ste50 in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Nusrat Sharmeen ◽  
Chris Law ◽  
Cunle Wu
Keyword(s):  
Cell Fate ◽  
Critical Role ◽  
Time Lapse ◽  
Yeast Cells ◽  
Cell Cortex ◽  
Dependent Manner ◽  
Pheromone Response ◽  
Concentration Dependent Manner ◽  
Directional Growth ◽  
Fate Decision

Polarization or directional growth is a major morphological change that occurs in yeast cells during pheromone response to mate with the opposite partner. In the pheromone signaling pathway, the adaptor Ste50 is required to bind MAP3K Ste11 for proper polarization; cells lacking Ste50 are impaired in polarization. Direct involvement of Ste50 in the polarization process has not been explored systematically. Here, we used single-cell fluorescent time-lapse microscopy to characterize Ste50 involvement in the establishment of cell polarity. We found early localization of Ste50 patches on the cell cortex that mark the point of shmoo initiation, these polarity sites move, and patches remain associated with the growing shmoo tip in a pheromone concentration-dependent manner until shmoo maturation. By quantitative analysis we show that polarization corelates with the rising levels of Ste50 enabling rapid individual cell responses to pheromone that corresponds to a critical level of Ste50 at the initial G1 phase. Suggesting Ste50 to be a pheromone responsive gene. We exploited the quantitative differences in the pattern of Ste50 expression to corelate with the cell-cell phenotypic heterogeneity showing Ste50 involvement in the cellular differentiation choices. Taken together, these findings present spatiotemporal localization of Ste50 during yeast polarization, suggesting that Ste50 is a component of the polarisome, and plays a critical role in regulating the polarized growth of shmoo during pheromone response.

scholarly journals The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro

2001 ◽  
Vol 154 (6) ◽  
pp. 1209-1224 ◽  
Author(s):  
Åsa E.Y. Engqvist-Goldstein ◽  
Robin A. Warren ◽  
Michael M. Kessels ◽  
James H. Keen ◽  
John Heuser ◽  
...  
Keyword(s):  
Binding Protein ◽  
Coiled Coil ◽  
Actin Binding ◽  
Live Cells ◽  
Cell Cortex ◽  
Coated Pits ◽  
Actin Binding Protein ◽  
Real Time Analysis

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R–YFP and DsRed–clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of ‘unroofed’ cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.

scholarly journals Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity

2014 ◽  
Vol 25 (9) ◽  
pp. 1421-1436 ◽  
Author(s):  
Jennifer M. Holden ◽  
Ludek Koreny ◽  
Samson Obado ◽  
Alexander V. Ratushny ◽  
Wei-Ming Chen ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Mitotic Spindle ◽  
Nuclear Periphery ◽  
Coiled Coil ◽  
Nuclear Lamina ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Major Barrier ◽  
African Trypanosomes

The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle–dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina.

scholarly journals Membrane Targeting and Asymmetric Localization of Drosophila Partner of Inscuteable Are Discrete Steps Controlled by Distinct Regions of the Protein

2002 ◽  
Vol 22 (12) ◽  
pp. 4230-4240 ◽  
Author(s):  
Fengwei Yu ◽  
Chin Tong Ong ◽  
William Chia ◽  
Xiaohang Yang
Keyword(s):  
Cell Fate ◽  
Mitotic Spindle ◽  
Protein Localization ◽  
Asymmetric Division ◽  
Terminal Region ◽  
Spindle Orientation ◽  
Functional Domain ◽  
Fate Determinants ◽  
Cell Fate Determinants ◽  
Apical Localization

ABSTRACT Asymmetric division of neural progenitors is a key mechanism by which neuronal diversity in the Drosophila central nervous system is generated. The distinct fates of the daughter cells derived from these divisions are achieved through preferential segregation of the cell fate determinants Prospero and Numb to one of the two daughters. This is achieved by coordinating apical and basal mitotic spindle orientation with the basal cortical localization of the cell fate determinants during mitosis. A complex of apically localized proteins, including Inscuteable (Insc), Partner of Inscuteable (Pins), Bazooka (Baz), DmPar-6, DaPKC, and Gαi, is required to mediate and coordinate basal protein localization with mitotic spindle orientation. Pins, a molecule which directly interacts with Insc, is a key component required for the integrity of this complex; in the absence of Pins, other components become mislocalized or destabilized, and basal protein localization and mitotic spindle orientation are defective. Here we define the functional domains of Pins. We show that the C-terminal region containing the Gαi binding GoLoco motifs is necessary and sufficient for targeting to the neuroblast cortex, which appears to be a prerequisite for apical localization of Pins. The N-terminal tetratricopeptide repeat-containing region of Pins is required for two processes; TPR repeats 1 to 3 plus the C-terminal region are required for apical localization but are insufficient to recruit Insc to the apical cortex, whereas TPR repeats 1 to 7 plus C-terminal Pins can perform both functions. Hence, the abilities of Pins to cortically localize, to apically localize, and to restore Insc apical localization are all separable, and all three capabilities are necessary to mediate asymmetric division. Moreover, the need for N-terminal Pins can be obviated by fusing a minimal Insc functional domain with the C-terminal region of Pins; this chimeric molecule is apically localized and can fulfill the functions of both Insc and Pins.

scholarly journals Tumor suppressor APC is an attenuator of spindle-pulling forces during C. elegans asymmetric cell division

2018 ◽  
Vol 115 (5) ◽  
pp. E954-E963 ◽  
Author(s):  
Kenji Sugioka ◽  
Lars-Eric Fielmich ◽  
Kota Mizumoto ◽  
Bruce Bowerman ◽  
Sander van den Heuvel ◽  
...  
Keyword(s):  
Cell Division ◽  
Tumor Suppressor ◽  
Mitotic Spindle ◽  
Asymmetric Cell Division ◽  
Cell Cortex ◽  
Dependent Manner ◽  
C Elegans ◽  
Pulling Forces ◽  
Underlying Mechanisms ◽  
Pulling Force

The adenomatous polyposis coli (APC) tumor suppressor has dual functions in Wnt/β-catenin signaling and accurate chromosome segregation and is frequently mutated in colorectal cancers. Although APC contributes to proper cell division, the underlying mechanisms remain poorly understood. Here we show that Caenorhabditis elegans APR-1/APC is an attenuator of the pulling forces acting on the mitotic spindle. During asymmetric cell division of the C. elegans zygote, a LIN-5/NuMA protein complex localizes dynein to the cell cortex to generate pulling forces on astral microtubules that position the mitotic spindle. We found that APR-1 localizes to the anterior cell cortex in a Par–aPKC polarity-dependent manner and suppresses anterior centrosome movements. Our combined cell biological and mathematical analyses support the conclusion that cortical APR-1 reduces force generation by stabilizing microtubule plus-ends at the cell cortex. Furthermore, APR-1 functions in coordination with LIN-5 phosphorylation to attenuate spindle-pulling forces. Our results document a physical basis for the attenuation of spindle-pulling force, which may be generally used in asymmetric cell division and, when disrupted, potentially contributes to division defects in cancer.

scholarly journals CLAMP/Spef1 regulates planar cell polarity signaling and asymmetric microtubule accumulation in the Xenopus ciliated epithelia

2018 ◽  
Vol 217 (5) ◽  
pp. 1633-1641 ◽  
Author(s):  
Sun K. Kim ◽  
Siwei Zhang ◽  
Michael E. Werner ◽  
Eva J. Brotslaw ◽  
Jennifer W. Mitchell ◽  
...  
Keyword(s):  
Cell Division ◽  
Epithelial Cells ◽  
Cell Signaling ◽  
Cell Polarity ◽  
Planar Cell Polarity ◽  
Protein Localization ◽  
Apical Cell ◽  
Cell Cortex ◽  
Pcp Signaling ◽  
Cell Cell

Most epithelial cells polarize along the axis of the tissue, a feature known as planar cell polarity (PCP). The initiation of PCP requires cell–cell signaling via the noncanonical Wnt/PCP pathway. Additionally, changes in the cytoskeleton both facilitate and reflect this polarity. We have identified CLAMP/Spef1 as a novel regulator of PCP signaling. In addition to decorating microtubules (MTs) and the ciliary rootlet, a pool of CLAMP localizes at the apical cell cortex. Depletion of CLAMP leads to the loss of PCP protein asymmetry, defects in cilia polarity, and defects in the angle of cell division. Additionally, depletion of CLAMP leads to a loss of the atypical cadherin-like molecule Celrs2, suggesting that CLAMP facilitates the stabilization of junctional interactions responsible for proper PCP protein localization. Depletion of CLAMP also affects the polarized organization of MTs. We hypothesize that CLAMP facilitates the establishment of cell polarity and promotes the asymmetric accumulation of MTs downstream of the establishment of proper PCP.

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