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An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region.

Schweizer N, Pawar N, Weiss M, Maiato H - J. Cell Biol. (2015)

Bottom Line: This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells.This membranous "spindle envelope" confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation.Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Chromosome Instability & Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal Cell Division Unit, Department of Experimental Biology, Faculdade de Medicina, Universidade do Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal.

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Soluble tubulin passively diffuses into thenuclearspace at NEB. (A) FRAP of GFP–α-tubulin in the nuclear region of mitotic S2 cells after MT depolymerization with colchicine. Bleaching was conducted at time 00:00 (minutes:seconds). The relative fluorescence (nuclear/cytoplasm) was set to 1 before bleaching. Data points represent means obtained from 10 cells, error bars represent standard deviations. Soluble tubulin is highly mobile and exchanges between compartments (t1/2 = 4.495 s; SD = 1.552 s; recovery = 98.5%; SD = 4.3%; n = 10 cells). (B) Laser microsurgery in an interphase S2 cell expressing Megator-mCherry and GFP–α-tubulin after MT depolymerization with colchicine. The arrowhead indicates the cut region. Membrane blebbing was occasionally observed in the cut region (arrow). Time is given in minutes:seconds, relative to laser microsurgery. Bars, 10 µm.
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fig2: Soluble tubulin passively diffuses into thenuclearspace at NEB. (A) FRAP of GFP–α-tubulin in the nuclear region of mitotic S2 cells after MT depolymerization with colchicine. Bleaching was conducted at time 00:00 (minutes:seconds). The relative fluorescence (nuclear/cytoplasm) was set to 1 before bleaching. Data points represent means obtained from 10 cells, error bars represent standard deviations. Soluble tubulin is highly mobile and exchanges between compartments (t1/2 = 4.495 s; SD = 1.552 s; recovery = 98.5%; SD = 4.3%; n = 10 cells). (B) Laser microsurgery in an interphase S2 cell expressing Megator-mCherry and GFP–α-tubulin after MT depolymerization with colchicine. The arrowhead indicates the cut region. Membrane blebbing was occasionally observed in the cut region (arrow). Time is given in minutes:seconds, relative to laser microsurgery. Bars, 10 µm.

Mentions: To analyze the dynamics of soluble tubulin in the nuclear region during early mitosis, we performed FRAP of GFP–α-tubulin in S2 cells, after MT depolymerization with colchicine at NEB. We found that after photobleaching the entire nuclear region, GFP–α-tubulin reaccumulated with a half-life of ∼4 s, with a percentage of recovery (nuclear/cytoplasmic ratio) close to 100% (Fig. 2 A). Thus, most if not all soluble tubulin in the nuclear region is mobile, in line with previous reports in sea urchin embryos (Salmon et al., 1984), but in contrast to a recently reported RanGTP-dependent immobile fraction in C. elegans (Hayashi et al., 2012).


An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region.

Schweizer N, Pawar N, Weiss M, Maiato H - J. Cell Biol. (2015)

Soluble tubulin passively diffuses into thenuclearspace at NEB. (A) FRAP of GFP–α-tubulin in the nuclear region of mitotic S2 cells after MT depolymerization with colchicine. Bleaching was conducted at time 00:00 (minutes:seconds). The relative fluorescence (nuclear/cytoplasm) was set to 1 before bleaching. Data points represent means obtained from 10 cells, error bars represent standard deviations. Soluble tubulin is highly mobile and exchanges between compartments (t1/2 = 4.495 s; SD = 1.552 s; recovery = 98.5%; SD = 4.3%; n = 10 cells). (B) Laser microsurgery in an interphase S2 cell expressing Megator-mCherry and GFP–α-tubulin after MT depolymerization with colchicine. The arrowhead indicates the cut region. Membrane blebbing was occasionally observed in the cut region (arrow). Time is given in minutes:seconds, relative to laser microsurgery. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4555823&req=5

fig2: Soluble tubulin passively diffuses into thenuclearspace at NEB. (A) FRAP of GFP–α-tubulin in the nuclear region of mitotic S2 cells after MT depolymerization with colchicine. Bleaching was conducted at time 00:00 (minutes:seconds). The relative fluorescence (nuclear/cytoplasm) was set to 1 before bleaching. Data points represent means obtained from 10 cells, error bars represent standard deviations. Soluble tubulin is highly mobile and exchanges between compartments (t1/2 = 4.495 s; SD = 1.552 s; recovery = 98.5%; SD = 4.3%; n = 10 cells). (B) Laser microsurgery in an interphase S2 cell expressing Megator-mCherry and GFP–α-tubulin after MT depolymerization with colchicine. The arrowhead indicates the cut region. Membrane blebbing was occasionally observed in the cut region (arrow). Time is given in minutes:seconds, relative to laser microsurgery. Bars, 10 µm.
Mentions: To analyze the dynamics of soluble tubulin in the nuclear region during early mitosis, we performed FRAP of GFP–α-tubulin in S2 cells, after MT depolymerization with colchicine at NEB. We found that after photobleaching the entire nuclear region, GFP–α-tubulin reaccumulated with a half-life of ∼4 s, with a percentage of recovery (nuclear/cytoplasmic ratio) close to 100% (Fig. 2 A). Thus, most if not all soluble tubulin in the nuclear region is mobile, in line with previous reports in sea urchin embryos (Salmon et al., 1984), but in contrast to a recently reported RanGTP-dependent immobile fraction in C. elegans (Hayashi et al., 2012).

Bottom Line: This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells.This membranous "spindle envelope" confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation.Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Chromosome Instability & Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal Cell Division Unit, Department of Experimental Biology, Faculdade de Medicina, Universidade do Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal.

Show MeSH
Related in: MedlinePlus