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Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

Giansanti MG, Vanderleest TE, Jewett CE, Sechi S, Frappaolo A, Fabian L, Robinett CC, Brill JA, Loerke D, Fuller MT, Blankenship JT - PLoS Genet. (2015)

Bottom Line: These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function.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.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Biologia e Biotecnologie, Università Sapienza di Roma, Roma, Italy.

ABSTRACT
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 in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun 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 onr and fun 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 onr and fun 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 onr and fun 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.

No MeSH data available.


Related in: MedlinePlus

Localization of exocyst complex proteins in dividing spermatocytes.(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.
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pgen.1005632.g002: Localization of exocyst complex proteins in dividing spermatocytes.(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.

Mentions: Localization of Sec8 protein was analyzed in primary spermatocytes from larval testes fixed with either methanol-free formaldehyde (Fig 2A) or methanol/formaldehyde (S2 Fig). Staining of interphase primary spermatocytes with anti-Tubulin and anti-Sec8 antibodies revealed that Sec8 protein was diffuse throughout the cytoplasm and enriched at the plasma membrane (Fig 2A). In dividing spermatocytes, in addition to localization at the plasma membrane, Sec8 was enriched in a broad cortical area at the cell equator and excluded from the poles (Fig 2A). During mid-telophase and late telophase, Sec8 protein accumulated at the cortex, near the ingressing furrow membrane (Fig 2A). Analysis of larval testes from transgenic animals expressing a GFP-Exo84 fusion protein revealed that, similar to Sec8, Exo84 appeared diffuse in the cytoplasm during interphase and became enriched in the furrow region during early telophase (S3 Fig). Analysis of dividing cells stained for Tubulin and Drosophila Sec5 revealed that Sec5 was enriched in small puncta at the astral microtubules and concentrated at the furrow region in telophase (Fig 2B).


Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

Giansanti MG, Vanderleest TE, Jewett CE, Sechi S, Frappaolo A, Fabian L, Robinett CC, Brill JA, Loerke D, Fuller MT, Blankenship JT - PLoS Genet. (2015)

Localization of exocyst complex proteins in dividing spermatocytes.(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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4631508&req=5

pgen.1005632.g002: Localization of exocyst complex proteins in dividing spermatocytes.(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.
Mentions: Localization of Sec8 protein was analyzed in primary spermatocytes from larval testes fixed with either methanol-free formaldehyde (Fig 2A) or methanol/formaldehyde (S2 Fig). Staining of interphase primary spermatocytes with anti-Tubulin and anti-Sec8 antibodies revealed that Sec8 protein was diffuse throughout the cytoplasm and enriched at the plasma membrane (Fig 2A). In dividing spermatocytes, in addition to localization at the plasma membrane, Sec8 was enriched in a broad cortical area at the cell equator and excluded from the poles (Fig 2A). During mid-telophase and late telophase, Sec8 protein accumulated at the cortex, near the ingressing furrow membrane (Fig 2A). Analysis of larval testes from transgenic animals expressing a GFP-Exo84 fusion protein revealed that, similar to Sec8, Exo84 appeared diffuse in the cytoplasm during interphase and became enriched in the furrow region during early telophase (S3 Fig). Analysis of dividing cells stained for Tubulin and Drosophila Sec5 revealed that Sec5 was enriched in small puncta at the astral microtubules and concentrated at the furrow region in telophase (Fig 2B).

Bottom Line: These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function.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.

View Article: PubMed Central - PubMed

Affiliation: Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Biologia e Biotecnologie, Università Sapienza di Roma, Roma, Italy.

ABSTRACT
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 in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun 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 onr and fun 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 onr and fun 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 onr and fun 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.

No MeSH data available.


Related in: MedlinePlus