scholarly journals Mitotic Spindle Positioning (MISP) is an actin bundler that selectively stabilizes the rootlets of epithelial microvilli

2021 ◽  
Author(s):  
E Angelo Morales ◽  
Cayetana Arnaiz ◽  
Evan S Krystofiak ◽  
Marija Zanic ◽  
Matthew J Tyska
Keyword(s):  
Mitotic Spindle ◽  
Apical Membrane ◽  
Membrane Surface ◽  
Actin Binding ◽  
Membrane Surface Area ◽  
Spindle Positioning ◽  
Cell Functions ◽  
The Core ◽  
Binding Factor

Microvilli are conserved actin-based surface protrusions that have been repurposed throughout evolution to fulfill diverse cell functions. In the case of transporting epithelia, microvilli are supported by a core of actin filaments bundled in parallel by villin, fimbrin, and espin. Remarkably, microvilli biogenesis persists in mice lacking all three of these factors, suggesting the existence of unknown bundlers. We identified Mitotic Spindle Positioning (MISP) as an actin binding factor that localizes specifically to the rootlet end of the microvillus. MISP promotes rootlet elongation in cells, and purified MISP exhibits potent filament bundling activity in vitro. MISP-bundled filaments also recruit fimbrin, which further elongates and stabilizes bundles. MISP confinement to the rootlet is enforced by ezrin, which prevents decoration of the membrane-wrapped distal end of the core bundle. These discoveries reveal how epithelial cells optimize apical membrane surface area and offer insight on the remarkable robustness of microvilli biogenesis.

scholarly journals Mitochondria-driven assembly of a cortical anchor for mitochondria and dynein

2017 ◽  
Vol 216 (10) ◽  
pp. 3061-3071 ◽  
Author(s):  
Lauren M. Kraft ◽  
Laura L. Lackner
Keyword(s):  
Plasma Membrane ◽  
Mitotic Spindle ◽  
Cell Cortex ◽  
Dual Function ◽  
Mitochondrial Inheritance ◽  
Cellular Functions ◽  
Core Component ◽  
Spindle Positioning ◽  
The Core ◽  
Contact Sites

Interorganelle contacts facilitate communication between organelles and impact fundamental cellular functions. In this study, we examine the assembly of the MECA (mitochondria–endoplasmic reticulum [ER]–cortex anchor), which tethers mitochondria to the ER and plasma membrane. We find that the assembly of Num1, the core component of MECA, requires mitochondria. Once assembled, Num1 clusters persistently anchor mitochondria to the cell cortex. Num1 clusters also function to anchor dynein to the plasma membrane, where dynein captures and walks along astral microtubules to help orient the mitotic spindle. We find that dynein is anchored by Num1 clusters that have been assembled by mitochondria. When mitochondrial inheritance is inhibited, Num1 clusters are not assembled in the bud, and defects in dynein-mediated spindle positioning are observed. The mitochondria-dependent assembly of a dual-function cortical anchor provides a mechanism to integrate the positioning and inheritance of the two essential organelles and expands the function of organelle contact sites.

High turnover of ezrin T567 phosphorylation: conformation, activity, and cellular function

2007 ◽  
Vol 293 (3) ◽  
pp. C874-C884 ◽  
Author(s):  
Lixin Zhu ◽  
Rihong Zhou ◽  
Shelley Mettler ◽  
Tim Wu ◽  
Aennes Abbas ◽  
...  
Keyword(s):  
Membrane Surface ◽  
Resonance Energy ◽  
Actin Binding ◽  
Live Cells ◽  
Filamentous Actin ◽  
High Turnover ◽  
Binding Conformation ◽  
Phosphorylated Form

In its dormant state, the membrane cytoskeletal linker protein ezrin takes on a NH2 terminal-to-COOH terminal (N-C) binding conformation. In vitro evidence suggests that eliminating the N-C binding conformation by Thr567 phosphorylation leads to ezrin activation. Here, we found for resting gastric parietal cells that the levels of ezrin phosphorylation on Thr567 are low and can be increased to a small extent (∼40%) by stimulating secretion via the cAMP pathway. Treatment of cells with protein phosphatase inhibitors led to a rapid, dramatic increase in Thr567 phosphorylation by 400% over resting levels, prompting the hypothesis that ezrin activity is regulated by turnover of phosphorylation on Thr567. In vitro and in vivo fluorescence resonance energy transfer analysis demonstrated that Thr567 phosphorylation opens the N-C interaction. However, even in the closed conformation, ezrin localizes to membranes by an exposed NH2 terminal binding site. Importantly, the opened phosphorylated form of ezrin more readily cosediments with F-actin and binds more tightly to membrane than the closed forms. Furthermore, fluorescence recovery after photobleaching analysis in live cells showed that the Thr567Asp mutant had longer recovery times than the wild type or the Thr567Ala mutant, indicating the Thr567-phosphorylated form of ezrin is tightly associated with F-actin and the membrane, restricting normal activity. These data demonstrate and emphasize the functional importance of reversible phosphorylation of ezrin on F-actin binding. A novel model is proposed whereby ezrin and closely associated kinase and phosphatase proteins represent a motor complex to maintain a dynamic relationship between the varying membrane surface area and filamentous actin length.

scholarly journals The enterocyte microvillus is a vesicle-generating organelle

2009 ◽  
Vol 185 (7) ◽  
pp. 1285-1298 ◽  
Author(s):  
Russell E. McConnell ◽  
James N. Higginbotham ◽  
David A. Shifrin ◽  
David L. Tabb ◽  
Robert J. Coffey ◽  
...  
Keyword(s):  
Brush Border ◽  
Membrane Surface ◽  
Intestinal Lumen ◽  
Intestinal Alkaline Phosphatase ◽  
Brush Border Enzymes ◽  
Membrane Surface Area ◽  
Catalytically Active ◽  
The Right

For decades, enterocyte brush border microvilli have been viewed as passive cytoskeletal scaffolds that serve to increase apical membrane surface area. However, recent studies revealed that in the in vitro context of isolated brush borders, myosin-1a (myo1a) powers the sliding of microvillar membrane along core actin bundles. This activity also leads to the shedding of small vesicles from microvillar tips, suggesting that microvilli may function as vesicle-generating organelles in vivo. In this study, we present data in support of this hypothesis, showing that enterocyte microvilli release unilamellar vesicles into the intestinal lumen; these vesicles retain the right side out orientation of microvillar membrane, contain catalytically active brush border enzymes, and are specifically enriched in intestinal alkaline phosphatase. Moreover, myo1a knockout mice demonstrate striking perturbations in vesicle production, clearly implicating this motor in the in vivo regulation of this novel activity. In combination, these data show that microvilli function as vesicle-generating organelles, which enable enterocytes to deploy catalytic activities into the intestinal lumen.

scholarly journals Microtubule end tethering of a processive kinesin-8 motor Kif18b is required for spindle positioning

2018 ◽  
Vol 217 (7) ◽  
pp. 2403-2416 ◽  
Author(s):  
Toni McHugh ◽  
Agata A. Gluszek ◽  
Julie P.I. Welburn
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Essential Role ◽  
Gene Knockout ◽  
Spindle Orientation ◽  
Binding Region ◽  
Spindle Positioning ◽  
Astral Microtubule ◽  
In Vitro Reconstitution

Mitotic spindle positioning specifies the plane of cell division during anaphase. Spindle orientation and positioning are therefore critical to ensure symmetric division in mitosis and asymmetric division during development. The control of astral microtubule length plays an essential role in positioning the spindle. In this study, using gene knockout, we show that the kinesin-8 Kif18b controls microtubule length to center the mitotic spindle at metaphase. Using in vitro reconstitution, we reveal that Kif18b is a highly processive plus end–directed motor that uses a C-terminal nonmotor microtubule-binding region to accumulate at growing microtubule plus ends. This region is regulated by phosphorylation to spatially control Kif18b accumulation at plus ends and is essential for Kif18b-dependent spindle positioning and regulation of microtubule length. Finally, we demonstrate that Kif18b shortens microtubules by increasing the catastrophe rate of dynamic microtubules. Overall, our work reveals that Kif18b uses its motile properties to reach microtubule ends, where it regulates astral microtubule length to ensure spindle centering.

scholarly journals Lasp1 gene disruption is linked to enhanced cell migration and tumor formation

2009 ◽  
Vol 38 (3) ◽  
pp. 372-385 ◽  
Author(s):  
Han Zhang ◽  
Xunsheng Chen ◽  
Wendy B. Bollag ◽  
Roni J. Bollag ◽  
Daniel J. Sheehan ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Lines ◽  
Focal Adhesion ◽  
Gene Disruption ◽  
Cancer Metastasis ◽  
Gene Knockout ◽  
Tumor Formation ◽  
Actin Binding ◽  
Cell Functions

Lasp1 is an actin-binding, signaling pathway-regulated phosphoprotein that is overexpressed in several cancers. siRNA knockdown in cell lines retards cell migration, suggesting the possibility that Lasp1 upregulation influences cancer metastasis. Herein, we utilized a recently developed gene knockout model to assess the role of Lasp1 in modulating nontransformed cell functions. Wound healing and tumor initiation progressed more rapidly in Lasp1−/− mice compared with Lasp1+/+ controls. Embryonic fibroblasts (MEFs) derived from Lasp1−/− mice also migrated more rapidly in vitro. These MEFs characteristically possessed increased focal adhesion numbers and displayed more rapid attachment compared with wild-type MEFs. Differential microarray analyses revealed alterations in message expression for proteins implicated in cell migration, adhesion, and cytoskeletal organization. Notably, the focal adhesion protein, lipoma preferred partner (LPP), a zyxin family member and putative Lasp1 binding protein, was increased about twofold. Because LPP gene disruption reduces cell migration, we hypothesize that LPP plays a role in enhancing the migratory capacity of Lasp1−/− MEFs, perhaps by modifying the subcellular localization of other motility-associated proteins. The striking contrast in the functional effects of loss of Lasp1 in innate cells compared with cell lines reveals distinct differences in mechanisms of motility and attachment in these models.

scholarly journals Optogenetic reconstitution reveals that Dynein-Dynactin-NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

2018 ◽  
Author(s):  
Masako Okumura ◽  
Toyoaki Natsume ◽  
Masato T. Kanemaki ◽  
Tomomi Kiyomitsu
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Human Cells ◽  
Microtubule Binding ◽  
Spindle Positioning ◽  
The Core ◽  
Pulling Forces ◽  
Focal Structures

AbstractTo position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment/activation by NuMA’s N-terminal long arm, and NuMA’s direct microtubule-binding activities to achieve a multiplicity of microtubule interactions. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.

scholarly journals Interaction of CK1δ with γTuSC ensures proper microtubule assembly and spindle positioning

2015 ◽  
Vol 26 (13) ◽  
pp. 2505-2518 ◽  
Author(s):  
Yutian Peng ◽  
Michelle Moritz ◽  
Xuemei Han ◽  
Thomas H. Giddings ◽  
Andrew Lyon ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Microtubule Assembly ◽  
Regulatory Mechanisms ◽  
Spindle Positioning ◽  
Small Complex ◽  
Binding Factor ◽  
Domain Loss ◽  
Cytoplasmic Microtubules

Casein kinase 1δ (CK1δ) family members associate with microtubule-organizing centers (MTOCs) from yeast to humans, but their mitotic roles and targets have yet to be identified. We show here that budding yeast CK1δ, Hrr25, is a γ-tubulin small complex (γTuSC) binding factor. Moreover, Hrr25's association with γTuSC depends on its kinase activity and its noncatalytic central domain. Loss of Hrr25 kinase activity resulted in assembly of unusually long cytoplasmic microtubules and defects in spindle positioning, consistent with roles in regulation of γTuSC-mediated microtubule nucleation and the Kar9 spindle-positioning pathway, respectively. Hrr25 directly phosphorylated γTuSC proteins in vivo and in vitro, and this phosphorylation promoted γTuSC integrity and activity. Because CK1δ and γTuSC are highly conserved and present at MTOCs in diverse eukaryotes, similar regulatory mechanisms are expected to apply generally in eukaryotes.

scholarly journals Dynein–Dynactin–NuMA clusters generate cortical spindle-pulling forces as a multi-arm ensemble

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Masako Okumura ◽  
Toyoaki Natsume ◽  
Masato T Kanemaki ◽  
Tomomi Kiyomitsu
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole ◽  
Human Cells ◽  
Microtubule Binding ◽  
Spindle Positioning ◽  
The Core ◽  
Astral Microtubule ◽  
Pulling Forces ◽  
Focal Structures

To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA’s N-terminal long arm, dynein-based astral microtubule gliding, and NuMA’s direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.

The everted renal tubule: a methodology for direct assessment of apical membrane function

1988 ◽  
Vol 255 (6) ◽  
pp. F1276-F1280 ◽  
Author(s):  
B. G. Engbretson ◽  
K. W. Beyenbach ◽  
L. C. Stoner
Keyword(s):  
Patch Clamp ◽  
Renal Tubule ◽  
Apical Membrane ◽  
Electrophysiological Study ◽  
Membrane Surface ◽  
Bathing Solution ◽  
Membrane Function ◽  
Large Conductance Channel ◽  
A Cell

A technique is described whereby it is possible to evert a 0.5- to 1.0-mm segment of an amphibian renal tubule and perfuse it in vitro. Consequently, the apical membranes of an intact nephron fragment are directly accessible for electrophysiological study. Viability of the cells of everted diluting segments taken from Ambystoma kidney was indicated by 1) failure of the cells to take up trypan blue and 2) the existence of an apical membrane voltage (average 66 mV, cell negative), which decreased predictably in the presence of either 5 mM barium or elevated potassium in the luminal bathing solution. The utility of the everted tubule to patch clamp studies was tested. A large conductance channel that appeared to be selective for potassium could be demonstrated in a cell-attached patch of the apical membrane of an everted initial collecting tubule. The everted tubule preparation not only provides large quantities of apical membrane for patch clamp studies but, more importantly, allows the investigator to control the solutions bathing each membrane surface independently. The application of patch clamp techniques to perfused, everted tubules may then serve to more completely describe the role of the apical membrane in transcellular ion transport.

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