PTEN controls glandular morphogenesis through a juxtamembrane β-Arrestin1/ARHGAP21 scaffolding complex

PTEN controls three-dimensional (3D) glandular morphogenesis by coupling juxtamembrane signaling to mitotic spindle machinery. While molecular mechanisms remain unclear, PTEN interacts through its C2 membrane-binding domain with the scaffold protein β-Arrestin1. Because β-Arrestin1 binds and suppresses the Cdc42 GTPase-activating protein ARHGAP21, we hypothesize that PTEN controls Cdc42 -dependent morphogenic processes through a β-Arrestin1-ARHGAP21 complex. Here, we show that PTEN knockdown (KD) impairs β-Arrestin1 membrane localization, β-Arrestin1-ARHGAP21 interactions, Cdc42 activation, mitotic spindle orientation and 3D glandular morphogenesis. Effects of PTEN deficiency were phenocopied by β-Arrestin1 KD or inhibition of β-Arrestin1-ARHGAP21 interactions. Conversely, silencing of ARHGAP21 enhanced Cdc42 activation and rescued aberrant morphogenic processes of PTEN-deficient cultures. Expression of the PTEN C2 domain mimicked effects of full-length PTEN but a membrane-binding defective mutant of the C2 domain abrogated these properties. Our results show that PTEN controls multicellular assembly through a membrane-associated regulatory protein complex composed of β-Arrestin1, ARHGAP21 and Cdc42.

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Aberrant endocytosis leads to the loss of normal mitotic spindle orientation during epithelial glandular morphogenesis

Journal of Biological Chemistry ◽

10.1074/jbc.ra117.001640 ◽

2018 ◽

Vol 293 (31) ◽

pp. 12095-12104

Author(s):

James W. Clancy ◽

Colin S. Sheehan ◽

Christopher J. Tricarico ◽

Crislyn D'Souza-Schorey

Keyword(s):

Mitotic Spindle ◽

Spindle Orientation ◽

Glandular Morphogenesis

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Plk1 regulates spindle orientation by phosphorylating NuMA in human cells

Life Science Alliance ◽

10.26508/lsa.201800223 ◽

2018 ◽

Vol 1 (6) ◽

pp. e201800223 ◽

Cited By ~ 14

Author(s):

Shrividya Sana ◽

Riya Keshri ◽

Ashwathi Rajeevan ◽

Sukriti Kapoor ◽

Sachin Kotak

Keyword(s):

Cell Division ◽

Mitotic Spindle ◽

Molecular Mechanisms ◽

Spindle Pole ◽

Spindle Orientation ◽

Cell Cortex ◽

Division Plane ◽

Evolutionarily Conserved ◽

Cortical Localization ◽

Proper Orientation

Proper orientation of the mitotic spindle defines the correct division plane and is essential for accurate cell division and development. In metazoans, an evolutionarily conserved complex comprising of NuMA/LGN/Gαi regulates proper orientation of the mitotic spindle by orchestrating cortical dynein levels during metaphase. However, the molecular mechanisms that modulate the spatiotemporal dynamics of this complex during mitosis remain elusive. Here, we report that acute inactivation of Polo-like kinase 1 (Plk1) during metaphase enriches cortical levels of dynein/NuMA/LGN and thus influences spindle orientation. We establish that this impact of Plk1 on cortical levels of dynein/NuMA/LGN is through NuMA, but not via dynein/LGN. Moreover, we reveal that Plk1 inhibition alters the dynamic behavior of NuMA at the cell cortex. We further show that Plk1 directly interacts and phosphorylates NuMA. Notably, NuMA-phosphorylation by Plk1 impacts its cortical localization, and this is needed for precise spindle orientation during metaphase. Overall, our finding connects spindle-pole pool of Plk1 with cortical NuMA and answers a long-standing puzzle about how spindle-pole Plk1 gradient dictates proper spindle orientation for error-free mitosis.

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A lateral belt of cortical LGN and NuMA guides mitotic spindle movements and planar division in neuroepithelial cells

The Journal of Cell Biology ◽

10.1083/jcb.201101039 ◽

2011 ◽

Vol 193 (1) ◽

pp. 141-154 ◽

Cited By ~ 97

Author(s):

Elise Peyre ◽

Florence Jaouen ◽

Mehdi Saadaoui ◽

Laurence Haren ◽

Andreas Merdes ◽

...

Keyword(s):

Mitotic Spindle ◽

Three Dimensional ◽

Active Phase ◽

Spindle Orientation ◽

Cell Cortex ◽

Neuroepithelial Cells ◽

Subsequent Phase ◽

Planar Orientation ◽

Lateral Cell ◽

Apical Localization

To maintain tissue architecture, epithelial cells divide in a planar fashion, perpendicular to their main polarity axis. As the centrosome resumes an apical localization in interphase, planar spindle orientation is reset at each cell cycle. We used three-dimensional live imaging of GFP-labeled centrosomes to investigate the dynamics of spindle orientation in chick neuroepithelial cells. The mitotic spindle displays stereotypic movements during metaphase, with an active phase of planar orientation and a subsequent phase of planar maintenance before anaphase. We describe the localization of the NuMA and LGN proteins in a belt at the lateral cell cortex during spindle orientation. Finally, we show that the complex formed of LGN, NuMA, and of cortically located Gαi subunits is necessary for spindle movements and regulates the dynamics of spindle orientation. The restricted localization of LGN and NuMA in the lateral belt is instructive for the planar alignment of the mitotic spindle, and required for its planar maintenance.

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Annexin A1 is a polarity cue that directs planar mitotic spindle orientation during mammalian epithelial morphogenesis

10.1101/2021.07.28.454117 ◽

2021 ◽

Author(s):

Maria Fankhaenel ◽

Farahnaz Sadat Golestan Hashemi ◽

Manal Mosa Hosawi ◽

Larissa Mourao ◽

Paul Skipp ◽

...

Keyword(s):

Mitotic Spindle ◽

Mammary Epithelial Cells ◽

Three Dimensional ◽

Mammary Epithelial ◽

Epithelial Morphogenesis ◽

Spindle Orientation ◽

Cell Cortex ◽

Annexin A1 ◽

Cell Divisions ◽

Cortical Patterning

Oriented cell divisions are critical for the formation and maintenance of structured epithelia. Proper mitotic spindle orientation relies on polarised anchoring of force generators to the cell cortex by the evolutionarily conserved Gαi-LGN-NuMA complex. However, the polarity cues that control cortical patterning of this ternary complex remain largely unknown in mammalian epithelia. Here we identify the membrane-associated protein Annexin A1 (ANXA1) as a novel interactor of LGN in mammary epithelial cells. ANXA1 acts independently of Gαi to instruct the accumulation of LGN and NuMA at the lateral cortex to ensure cortical anchoring of Dynein-Dynactin and astral microtubules and thereby planar alignment of the mitotic spindle. Loss of ANXA1 randomises mitotic spindle orientation, which in turn disrupts epithelial architecture and lumen formation in three-dimensional (3D) primary mammary organoids. Our findings establish ANXA1 as an upstream cortical cue that regulates LGN to direct planar cell divisions during mammalian epithelial morphogenesis.

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Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158376 ◽

1990 ◽

Vol 48 (3) ◽

pp. 166-167

Author(s):

J. Holy ◽

G. Schatten

Keyword(s):

Confocal Microscopy ◽

Mitotic Spindle ◽

Laser Scanning ◽

Three Dimensional ◽

Laser Scanning Confocal Microscopy ◽

Two Dimensions ◽

Embryonic Cells ◽

Mitotic Spindles ◽

Cleavage Division ◽

Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

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Structural Studies of Fibrinolysis by Electron and Light Microscopy

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615843 ◽

1999 ◽

Vol 82 (08) ◽

pp. 277-282 ◽

Cited By ~ 37

Author(s):

Yuri Veklich ◽

Jean-Philippe Collet ◽

Charles Francis ◽

John W. Weisel

Keyword(s):

Electron Microscopy ◽

Light Microscopy ◽

Plasminogen Activator ◽

Structural Changes ◽

Molecular Mechanisms ◽

Degradation Products ◽

Molecular Model ◽

Three Dimensional ◽

Tissue Type ◽

Type Plasminogen Activator

IntroductionMuch is known about the fibrinolytic system that converts fibrin-bound plasminogen to the active protease, plasmin, using plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator. Plasmin then cleaves fibrin at specific sites and generates soluble fragments, many of which have been characterized, providing the basis for a molecular model of the polypeptide chain degradation.1-3 Soluble degradation products of fibrin have also been characterized by transmission electron microscopy, yielding a model for their structure.4 Moreover, high resolution, three-dimensional structures of certain fibrinogen fragments has provided a wealth of information that may be useful in understanding how various proteins bind to fibrin and the overall process of fibrinolysis (Doolittle, this volume).5,6 Both the rate of fibrinolysis and the structures of soluble derivatives are determined in part by the fibrin network structure itself. Furthermore, the activation of plasminogen by t-PA is accelerated by the conversion of fibrinogen to fibrin, and this reaction is also affected by the structure of the fibrin. For example, clots made of thin fibers have a decreased rate of conversion of plasminogen to plasmin by t-PA, and they generally are lysed more slowly than clots composed of thick fibers.7-9 Under other conditions, however, clots made of thin fibers may be lysed more rapidly.10 In addition, fibrin clots composed of abnormally thin fibers formed from certain dysfibrinogens display decreased plasminogen binding and a lower rate of fibrinolysis.11-13 Therefore, our increasing knowledge of various dysfibrinogenemias will aid our understanding of mechanisms of fibrinolysis (Matsuda, this volume).14,15 To account for these diverse observations and more fully understand the molecular basis of fibrinolysis, more knowledge of the physical changes in the fibrin matrix that precede solubilization is required. In this report, we summarize recent experiments utilizing transmission and scanning electron microscopy and confocal light microscopy to provide information about the structural changes occurring in polymerized fibrin during fibrinolysis. Many of the results of these experiments were unexpected and suggest some aspects of potential molecular mechanisms of fibrinolysis, which will also be described here.

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