Trisomy 21 induces pericentrosomal crowding delaying primary ciliogenesis and mouse cerebellar development.

Trisomy 21, the genetic cause of Down syndrome, disrupts primary cilia formation and function, in part through elevated Pericentrin, a centrosome protein encoded on chromosome 21. Yet how trisomy 21 and elevated Pericentrin disrupt cilia-related molecules and pathways, and the in vivo phenotypic relevance remain unclear. Utilizing ciliogenesis time course experiments combined with light microscopy and electron tomography, we reveal that chromosome 21 polyploidy elevates Pericentrin and microtubules away from the centrosome that corral MyosinVA and EHD1, delaying ciliary membrane delivery and mother centriole uncapping essential for ciliogenesis. If given enough time, trisomy 21 cells eventually ciliate, but these ciliated cells demonstrate persistent trafficking defects that reduce transition zone protein localization and decrease sonic hedgehog signaling in direct anticorrelation with Pericentrin levels. Consistent with cultured trisomy 21 cells, a mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers at P4. Our work reveals that elevated Pericentrin from trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding mechanism results in signaling deficiencies consistent with the neurological phenotypes found in individuals with Down syndrome

Cell Ratcheting through the Sbf RabGEF Directs Force Balancing and Stepped Apical Constriction

During Drosophila melanogaster gastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a trafficking pathway initiated by Sbf is disrupted, contractile steps become reversible. Sbf localizes to tubular, apical surfaces and associates with Rab35, where it promotes Rab GTP exchange. Interestingly, when Sbf/Rab35 function is compromised, the apical plasma membrane becomes deeply convoluted, and nonuniform cell behaviors begin to emerge. Consistent with this, Sbf/Rab35 appears to prefigure and organize the apical surface for efficient Myosin function. Finally, we show that Sbf/Rab35/CME directs the plasma membrane to Rab11 endosomes through a dynamic interaction with Rab5 endosomes. These results suggest that periodic ratcheting events shift excess membrane from cell apices into endosomal pathways to permit reshaping of actomyosin networks and the apical surface

Vertex Sliding Drives Intercalation by Radial Coupling of Adhesion and Actomyosin Networks during Drosophila Germband Extension

Oriented cell intercalation is an essential developmental process that shapes tissue morphologies through the directional insertion of cells between their neighbors. Previous research has focused on properties of cell–cell interfaces, while the function of tricellular vertices has remained unaddressed. Here, we identify a highly novel mechanism in which vertices demonstrate independent sliding behaviors along cell peripheries to produce the topological deformations responsible for intercalation. Through systematic analysis, we find that the motion of vertices connected by contracting interfaces is not physically coupled, but instead possess strong radial coupling. E-cadherin and Myosin II exist in previously unstudied populations at cell vertices and undergo oscillatory cycles of accumulation and dispersion that are coordinated with changes in cell area. Additionally, peak enrichment of vertex E-cadherin/Myosin II coincides with interface length stabilization. Our results suggest a model in which asymmetric radial force balance directs the progressive, ratcheted motion of individual vertices to drive intercalation

Vertex Sliding Drives Intercalation by Radial Coupling of Adhesion and Actomyosin Networks during Drosophila Germband Extension

Oriented cell intercalation is an essential developmental process that shapes tissue morphologies through the directional insertion of cells between their neighbors. Previous research has focused on properties of cell–cell interfaces, while the function of tricellular vertices has remained unaddressed. Here, we identify a highly novel mechanism in which vertices demonstrate independent sliding behaviors along cell peripheries to produce the topological deformations responsible for intercalation. Through systematic analysis, we find that the motion of vertices connected by contracting interfaces is not physically coupled, but instead possess strong radial coupling. E-cadherin and Myosin II exist in previously unstudied populations at cell vertices and undergo oscillatory cycles of accumulation and dispersion that are coordinated with changes in cell area. Additionally, peak enrichment of vertex E-cadherin/Myosin II coincides with interface length stabilization. Our results suggest a model in which asymmetric radial force balance directs the progressive, ratcheted motion of individual vertices to drive intercalation

Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in <i>Drosophila</i>

<div><p>Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division <i>in vivo</i>, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that <i>onion rings (onr)</i> and <i>funnel cakes (fun)</i> encode the <i>Drosophila</i> homologs of the Exo84 and Sec8 exocyst subunits, respectively. In <i>onr</i> and <i>fun</i> mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus <i>onr</i> and <i>fun</i> mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that <i>onr</i> and <i>fun</i> mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in <i>onr</i> and <i>fun</i> spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.</p></div

Cell ratcheting through the Sbf RabGEF directs force balancing and stepped apical constriction

During Drosophila melanogaster gastrulation, the invagination of the prospective mesoderm is driven by the pulsed constriction of apical surfaces. Here, we address the mechanisms by which the irreversibility of pulsed events is achieved while also permitting uniform epithelial behaviors to emerge. We use MSD-based analyses to identify contractile steps and find that when a trafficking pathway initiated by Sbf is disrupted, contractile steps become reversible. Sbf localizes to tubular, apical surfaces and associates with Rab35, where it promotes Rab GTP exchange. Interestingly, when Sbf/Rab35 function is compromised, the apical plasma membrane becomes deeply convoluted, and nonuniform cell behaviors begin to emerge. Consistent with this, Sbf/Rab35 appears to prefigure and organize the apical surface for efficient Myosin function. Finally, we show that Sbf/Rab35/CME directs the plasma membrane to Rab11 endosomes through a dynamic interaction with Rab5 endosomes. These results suggest that periodic ratcheting events shift excess membrane from cell apices into endosomal pathways to permit reshaping of actomyosin networks and the apical surface

<i>onr</i> and <i>fun</i> mutations interact with mutations in <i>Rab11</i>.

<p>(A) Frequencies of early spermatids containing 2, 4 or more than 4 nuclei per nebenkern in testes from either <i>Rab11</i>^<i>93Bi</i><i>/Rab11</i>^<i>93Bi</i><i>(Rab11) or fun</i>^<i>z1010</i><i>Rab11</i>^<i>93Bi</i><i>/+ Rab11</i>^<i>93Bi</i><i>(fun Rab11/Rab11)</i> mutant males. (B) Frequencies of early spermatids containing multiple nuclei (2, 4 or more than 4 nuclei) per nebenkern in testes from either <i>Rab11</i>^<i>93Bi</i>/<i>Rab11</i>^{<i>E(To)3</i>}<i>(Rab11)</i>, <i>fun</i>^<i>z1010</i><i>/fun</i>^<i>z1010</i><i>(fun)</i>, or <i>fun</i>^<i>z1010</i><i>Rab11</i>^<i>93Bi</i><i>fun</i>^<i>z1010</i><i>Rab11</i>^{<i>E(To)3</i>}<i>(fun Rab11)</i> mutant males. (C) Co-IP of HA-Sec8 with GFP-Exo84. Protein extracts from testes expressing either HA-Sec8 and GFP-Exo84 or HA-Sec8 alone were immunoprecipitated with anti-GFP (i.e., GFP-trap beads) and immunoblotted for either GFP, HA or Rab11. (D) Co-IP of Sec5 with YFP-Rab11. Protein extracts from testes expressing either wild-type YFP-Rab11 (wt), YFP-Rab11^Q70L (Q70L) or YFP-Rab11^S25N (S25N) were immunoprecipitated for YFP (using GFP-trap beads) and blotted for either YFP or Sec5.</p

Defective cytokinetic ring ingression in <i>fun</i> and <i>onr</i> mutant cells.

<p>(A) Selected still frames from supplemental <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005632#pgen.1005632.s005" target="_blank">S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005632#pgen.1005632.s006" target="_blank">S2</a></b>and <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005632#pgen.1005632.s007" target="_blank">S3</a></b>Movies. Dividing spermatocytes expressing the regulatory light chain of non-muscle myosin II, Sqh-GFP, were imaged starting from the beginning of anaphase. Numbers at the bottom of each frame indicate minutes from the beginning of imaging. Note that the Sqh-GFP ring undergoes minimal constriction (<i>fun</i>) or fails to constrict (<i>onr</i>) in mutant cells. Scale bar, 10μm. (B) Dynamics of cleavage furrows in <i>fun</i> and <i>onr</i> mutants. Furrow diameters (relative to the diameter at t = 0) in dividing spermatocytes from wild type, <i>fun</i>^<i>z1010</i><i>/Df(3R)Exel6145</i> (<i>fun</i>) and <i>onr</i>^<i>z4840</i><i>/Df(3R)Espl1 (onr)</i> males expressing Sqh-GFP and undergoing ana-telophase were plotted over time. (C) Furrow diameters (relative to the diameter at time = 0) were plotted at 5-minute intervals. Furrow diameters were measured in movies from dividing spermatocytes expressing Sqh-GFP and undergoing ana-telophases (n = 9 wild type, n = 8 <i>fun</i> and n = 8 <i>onr)</i>. Error bars indicate standard deviations. *p = 0.0035, **p = 0.0008;***p = 0.0001, significantly different from control in the Student t test.</p

Localization of exocyst complex proteins in dividing spermatocytes.

<p>(A) Localization of Sec8 protein in wild-type primary spermatocytes. Interphase and dividing spermatocytes were stained for Tubulin (green), Sec8 (red) and DNA (blue). During interphase, Sec8 was mostly diffuse in the cytoplasm and enriched at the plasma membrane (arrowheads). In dividing spermatocytes, Sec8 appeared enriched in a broad cortical band that encircled the midzone (arrows) and was excluded from the poles. (B) Localization of Sec5 protein in wild-type dividing spermatocytes. Primary spermatocytes were stained for Tubulin (green), Sec5 (red) and DNA (blue). Note the enrichment of Sec5 in puncta at the astral microtubules (arrowhead) and at the cleavage furrow (arrows). Scale bar, 10 μm.</p