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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

onr and fun mutations interact with mutations in Rab11.(A) Frequencies of early spermatids containing 2, 4 or more than 4 nuclei per nebenkern in testes from either Rab1193Bi/Rab1193Bi(Rab11) or funz1010Rab1193Bi/+ Rab1193Bi(fun Rab11/Rab11) mutant males. (B) Frequencies of early spermatids containing multiple nuclei (2, 4 or more than 4 nuclei) per nebenkern in testes from either Rab1193Bi/Rab11E(To)3(Rab11), funz1010/funz1010(fun), or funz1010Rab1193Bifunz1010Rab11E(To)3(fun Rab11) 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-Rab11Q70L (Q70L) or YFP-Rab11S25N (S25N) were immunoprecipitated for YFP (using GFP-trap beads) and blotted for either YFP or Sec5.
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pgen.1005632.g009: onr and fun mutations interact with mutations in Rab11.(A) Frequencies of early spermatids containing 2, 4 or more than 4 nuclei per nebenkern in testes from either Rab1193Bi/Rab1193Bi(Rab11) or funz1010Rab1193Bi/+ Rab1193Bi(fun Rab11/Rab11) mutant males. (B) Frequencies of early spermatids containing multiple nuclei (2, 4 or more than 4 nuclei) per nebenkern in testes from either Rab1193Bi/Rab11E(To)3(Rab11), funz1010/funz1010(fun), or funz1010Rab1193Bifunz1010Rab11E(To)3(fun Rab11) 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-Rab11Q70L (Q70L) or YFP-Rab11S25N (S25N) were immunoprecipitated for YFP (using GFP-trap beads) and blotted for either YFP or Sec5.

Mentions: The onr and fun mutants interacted genetically with Rab11 mutants. Heterozygosity for fun dramatically increased the frequency of cytokinesis failures caused by homozygosity for the weak Rab11 allele Rab1193Bi, indicating a strong genetic interaction. funz1010Rab1193Bi/+Rab1193Bi males raised at 25°C exhibited a 7-fold increase in the percentage of multinucleate spermatids relative to testes from Rab1193Bi/Rab1193Bi single mutants (Fig 9A and 9B). In addition, although Rab1193Bi and Rab1193Bi/Rab11E(To)11 transheterozygotes were viable, as were funz1010/ funz1010 flies, funz1010Rab1193Bi/ funz1010Rab11E(To)11 double mutants died mostly at early larval stages. Examination of testes from rare escaper larvae of genotype funz1010Rab1193Bi/ funz1010Rab11E(To)11 revealed that 13.9% of spermatids exhibited more than four nuclei per mitochondrial derivative, indicating a dramatic increase in cytokinesis failures during the gonial divisions that precede meiosis (Fig 9B). Rab11 also interacted genetically with onr. onrz4840Rab1193Bi double mutants died in early larval stages, as did individuals that were homozygous for onrz4840 and transheterozygous for Rab1193Bi/Rab11E(To)11.


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)

onr and fun mutations interact with mutations in Rab11.(A) Frequencies of early spermatids containing 2, 4 or more than 4 nuclei per nebenkern in testes from either Rab1193Bi/Rab1193Bi(Rab11) or funz1010Rab1193Bi/+ Rab1193Bi(fun Rab11/Rab11) mutant males. (B) Frequencies of early spermatids containing multiple nuclei (2, 4 or more than 4 nuclei) per nebenkern in testes from either Rab1193Bi/Rab11E(To)3(Rab11), funz1010/funz1010(fun), or funz1010Rab1193Bifunz1010Rab11E(To)3(fun Rab11) 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-Rab11Q70L (Q70L) or YFP-Rab11S25N (S25N) were immunoprecipitated for YFP (using GFP-trap beads) and blotted for either YFP or Sec5.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4631508&req=5

pgen.1005632.g009: onr and fun mutations interact with mutations in Rab11.(A) Frequencies of early spermatids containing 2, 4 or more than 4 nuclei per nebenkern in testes from either Rab1193Bi/Rab1193Bi(Rab11) or funz1010Rab1193Bi/+ Rab1193Bi(fun Rab11/Rab11) mutant males. (B) Frequencies of early spermatids containing multiple nuclei (2, 4 or more than 4 nuclei) per nebenkern in testes from either Rab1193Bi/Rab11E(To)3(Rab11), funz1010/funz1010(fun), or funz1010Rab1193Bifunz1010Rab11E(To)3(fun Rab11) 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-Rab11Q70L (Q70L) or YFP-Rab11S25N (S25N) were immunoprecipitated for YFP (using GFP-trap beads) and blotted for either YFP or Sec5.
Mentions: The onr and fun mutants interacted genetically with Rab11 mutants. Heterozygosity for fun dramatically increased the frequency of cytokinesis failures caused by homozygosity for the weak Rab11 allele Rab1193Bi, indicating a strong genetic interaction. funz1010Rab1193Bi/+Rab1193Bi males raised at 25°C exhibited a 7-fold increase in the percentage of multinucleate spermatids relative to testes from Rab1193Bi/Rab1193Bi single mutants (Fig 9A and 9B). In addition, although Rab1193Bi and Rab1193Bi/Rab11E(To)11 transheterozygotes were viable, as were funz1010/ funz1010 flies, funz1010Rab1193Bi/ funz1010Rab11E(To)11 double mutants died mostly at early larval stages. Examination of testes from rare escaper larvae of genotype funz1010Rab1193Bi/ funz1010Rab11E(To)11 revealed that 13.9% of spermatids exhibited more than four nuclei per mitochondrial derivative, indicating a dramatic increase in cytokinesis failures during the gonial divisions that precede meiosis (Fig 9B). Rab11 also interacted genetically with onr. onrz4840Rab1193Bi double mutants died in early larval stages, as did individuals that were homozygous for onrz4840 and transheterozygous for Rab1193Bi/Rab11E(To)11.

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