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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

Rescue of funnel cakes and onion rings mutant cells by Sec8 and Exo84.(A) Phase contrast microscopy of wild type, funz1010/Df(3R)Exel6145 mutant (B) and onrz4840/Df(3R)Espl3 mutant (C) male germline cells. In fun and onr mutant cells, cell division fails and multiple nuclei (white spherical objects) are observed in association with enlarged nebenkern (black spherical objects). In wild-type cells, single nuclei are found in association with nebenkern of approximately equal size. A single copy of a transgene containing either genomic Sec8 (D) or genomic Exo84 (E) rescues cytokinesis defects in funz1010/Df(3R)Exel6145 (D) and onrz4840/Df(3R)Espl3 (E) mutant cells. Scale bar, 10 μm.
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pgen.1005632.g001: Rescue of funnel cakes and onion rings mutant cells by Sec8 and Exo84.(A) Phase contrast microscopy of wild type, funz1010/Df(3R)Exel6145 mutant (B) and onrz4840/Df(3R)Espl3 mutant (C) male germline cells. In fun and onr mutant cells, cell division fails and multiple nuclei (white spherical objects) are observed in association with enlarged nebenkern (black spherical objects). In wild-type cells, single nuclei are found in association with nebenkern of approximately equal size. A single copy of a transgene containing either genomic Sec8 (D) or genomic Exo84 (E) rescues cytokinesis defects in funz1010/Df(3R)Exel6145 (D) and onrz4840/Df(3R)Espl3 (E) mutant cells. Scale bar, 10 μm.

Mentions: fun and onr were identified in a screen for mutations that disrupt cytokinetic events in male germline cells [4]. Previous characterization of fun and onr revealed that these mutations do not affect central spindle or F-actin ring formation in dividing spermatocytes. Nonetheless, in fun and onr mutants, cytokinesis fails at an early stage [4]. The funz1010 mutation was mapped to the 83C1;83C4 interval on chromosome III in the region of the Sec8 gene. Deficiency mapping revealed that funz1010 failed to complement Df(3R)Exel6145 for the male sterility and cytokinesis defects (Fig 1A and 1B). Two lines of evidence indicate that funz1010 is an allele of Drosophila Sec8, which encodes a protein with 35% identity to human and mouse Sec8 proteins and 19% identity to the S. cerevisiae Sec8 protein (S1 Fig). First, a 6.6 kb genomic transgene containing the predicted Sec8 coding region, 1.0 kb of upstream promoter sequence, and 1.9 kb of downstream sequence fully rescued the cytokinesis defects in fun mutant male germline cells (Fig 1B and 1D). Indeed, 100% of onion-stage spermatids from funz1010/Df(3R)Exel6145 males bearing a single copy of the rescuing transgene possess a wild type 1:1 ratio of nuclei to nebenkern (n = 102), compared with 0.8% in males of identical genotype devoid of the transgene (n = 125). Additionally, DNA sequencing of the Sec8 gene in funz1010 mutant males revealed a C to T mutation resulting in replacement of a conserved Serine residue by Phenylalanine at position 322 of the predicted 985 amino acid polypeptide (S1 Fig). Together, these results provide clear evidence that funz1010 represents a mutation in the Sec8 gene.


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)

Rescue of funnel cakes and onion rings mutant cells by Sec8 and Exo84.(A) Phase contrast microscopy of wild type, funz1010/Df(3R)Exel6145 mutant (B) and onrz4840/Df(3R)Espl3 mutant (C) male germline cells. In fun and onr mutant cells, cell division fails and multiple nuclei (white spherical objects) are observed in association with enlarged nebenkern (black spherical objects). In wild-type cells, single nuclei are found in association with nebenkern of approximately equal size. A single copy of a transgene containing either genomic Sec8 (D) or genomic Exo84 (E) rescues cytokinesis defects in funz1010/Df(3R)Exel6145 (D) and onrz4840/Df(3R)Espl3 (E) mutant cells. Scale bar, 10 μm.
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pgen.1005632.g001: Rescue of funnel cakes and onion rings mutant cells by Sec8 and Exo84.(A) Phase contrast microscopy of wild type, funz1010/Df(3R)Exel6145 mutant (B) and onrz4840/Df(3R)Espl3 mutant (C) male germline cells. In fun and onr mutant cells, cell division fails and multiple nuclei (white spherical objects) are observed in association with enlarged nebenkern (black spherical objects). In wild-type cells, single nuclei are found in association with nebenkern of approximately equal size. A single copy of a transgene containing either genomic Sec8 (D) or genomic Exo84 (E) rescues cytokinesis defects in funz1010/Df(3R)Exel6145 (D) and onrz4840/Df(3R)Espl3 (E) mutant cells. Scale bar, 10 μm.
Mentions: fun and onr were identified in a screen for mutations that disrupt cytokinetic events in male germline cells [4]. Previous characterization of fun and onr revealed that these mutations do not affect central spindle or F-actin ring formation in dividing spermatocytes. Nonetheless, in fun and onr mutants, cytokinesis fails at an early stage [4]. The funz1010 mutation was mapped to the 83C1;83C4 interval on chromosome III in the region of the Sec8 gene. Deficiency mapping revealed that funz1010 failed to complement Df(3R)Exel6145 for the male sterility and cytokinesis defects (Fig 1A and 1B). Two lines of evidence indicate that funz1010 is an allele of Drosophila Sec8, which encodes a protein with 35% identity to human and mouse Sec8 proteins and 19% identity to the S. cerevisiae Sec8 protein (S1 Fig). First, a 6.6 kb genomic transgene containing the predicted Sec8 coding region, 1.0 kb of upstream promoter sequence, and 1.9 kb of downstream sequence fully rescued the cytokinesis defects in fun mutant male germline cells (Fig 1B and 1D). Indeed, 100% of onion-stage spermatids from funz1010/Df(3R)Exel6145 males bearing a single copy of the rescuing transgene possess a wild type 1:1 ratio of nuclei to nebenkern (n = 102), compared with 0.8% in males of identical genotype devoid of the transgene (n = 125). Additionally, DNA sequencing of the Sec8 gene in funz1010 mutant males revealed a C to T mutation resulting in replacement of a conserved Serine residue by Phenylalanine at position 322 of the predicted 985 amino acid polypeptide (S1 Fig). Together, these results provide clear evidence that funz1010 represents a mutation in the Sec8 gene.

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