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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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Protein accumulation in the nuclear/spindle region is a consequence of different molecular crowding states. (A) Cells with different diameters (λ) and different spatial positions of the FCS measurements were made comparable by expressing measurement positions relative to the cell size (red and orange bullets). Dashed circles indicate the nuclear space; gray-shaded objects symbolize condensed chromatin. (B) FCS analysis of GFP–α-tubulin, Megator-mCherry, and mRFP-Mad2 during mitosis after MT depolymerization with colchicine. Depicted are the mean or median values of the dwell time τD and the anomaly degree (α) of the diffusional motion within the focus in both the nuclear region (N) and cytoplasm (C). ***, P < 0.001. (C–E) FCS measurements were performed at 10 loci per cell (tubulin, 11 cells; Megator, 15 cells; Mad2, 15 cells). Position data were rescaled (nuclear region: λ = −0.2, . . . , +0.2). The number of fluorescent molecules (N) within the focus was relativized to the maximum number of fluorescent molecules (Nmax) detected in each cell to account for varying expression levels. (F and G) Modeling of protein diffusion into/out of the nuclear region. Diffusion of soluble tubulin (F) into and Mad2 (G) out of the nuclear region progresses within few seconds to a steady state, as indicated by the apparent fluorescence in the nuclear region Fn(t) and the cytoplasm Fc(t). Representative fluorescence images of the initial condition, an intermediate state, and the steady state are shown in the top panels. Dashed white lines indicate the respective line scans used for quantification of fluorescence (inset graphs).
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fig4: Protein accumulation in the nuclear/spindle region is a consequence of different molecular crowding states. (A) Cells with different diameters (λ) and different spatial positions of the FCS measurements were made comparable by expressing measurement positions relative to the cell size (red and orange bullets). Dashed circles indicate the nuclear space; gray-shaded objects symbolize condensed chromatin. (B) FCS analysis of GFP–α-tubulin, Megator-mCherry, and mRFP-Mad2 during mitosis after MT depolymerization with colchicine. Depicted are the mean or median values of the dwell time τD and the anomaly degree (α) of the diffusional motion within the focus in both the nuclear region (N) and cytoplasm (C). ***, P < 0.001. (C–E) FCS measurements were performed at 10 loci per cell (tubulin, 11 cells; Megator, 15 cells; Mad2, 15 cells). Position data were rescaled (nuclear region: λ = −0.2, . . . , +0.2). The number of fluorescent molecules (N) within the focus was relativized to the maximum number of fluorescent molecules (Nmax) detected in each cell to account for varying expression levels. (F and G) Modeling of protein diffusion into/out of the nuclear region. Diffusion of soluble tubulin (F) into and Mad2 (G) out of the nuclear region progresses within few seconds to a steady state, as indicated by the apparent fluorescence in the nuclear region Fn(t) and the cytoplasm Fc(t). Representative fluorescence images of the initial condition, an intermediate state, and the steady state are shown in the top panels. Dashed white lines indicate the respective line scans used for quantification of fluorescence (inset graphs).

Mentions: To investigate how a fenestrated organelle-exclusion envelope drives protein accumulation in the nuclear/spindle region during early mitosis, we characterized the diffusion properties of GFP–α-tubulin and Megator-mCherry at a single-molecule level using fluorescence correlation spectroscopy (FCS) in S2 cells. To ensure the independence from MTs, they were depolymerized with colchicine. While soluble tubulin showed similar anomalous diffusion inside and outside the nuclear region, Megator diffusion was about five times slower in the nuclear region when compared with the rest of the cytoplasm (Fig. 4, A–E), where Megator diffused more anomalously (i.e., more obstructed). These differences might reflect interactions with at least two other nuclear-derived proteins: Chromator and EAST (Qi et al., 2004, 2005).


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)

Protein accumulation in the nuclear/spindle region is a consequence of different molecular crowding states. (A) Cells with different diameters (λ) and different spatial positions of the FCS measurements were made comparable by expressing measurement positions relative to the cell size (red and orange bullets). Dashed circles indicate the nuclear space; gray-shaded objects symbolize condensed chromatin. (B) FCS analysis of GFP–α-tubulin, Megator-mCherry, and mRFP-Mad2 during mitosis after MT depolymerization with colchicine. Depicted are the mean or median values of the dwell time τD and the anomaly degree (α) of the diffusional motion within the focus in both the nuclear region (N) and cytoplasm (C). ***, P < 0.001. (C–E) FCS measurements were performed at 10 loci per cell (tubulin, 11 cells; Megator, 15 cells; Mad2, 15 cells). Position data were rescaled (nuclear region: λ = −0.2, . . . , +0.2). The number of fluorescent molecules (N) within the focus was relativized to the maximum number of fluorescent molecules (Nmax) detected in each cell to account for varying expression levels. (F and G) Modeling of protein diffusion into/out of the nuclear region. Diffusion of soluble tubulin (F) into and Mad2 (G) out of the nuclear region progresses within few seconds to a steady state, as indicated by the apparent fluorescence in the nuclear region Fn(t) and the cytoplasm Fc(t). Representative fluorescence images of the initial condition, an intermediate state, and the steady state are shown in the top panels. Dashed white lines indicate the respective line scans used for quantification of fluorescence (inset graphs).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Protein accumulation in the nuclear/spindle region is a consequence of different molecular crowding states. (A) Cells with different diameters (λ) and different spatial positions of the FCS measurements were made comparable by expressing measurement positions relative to the cell size (red and orange bullets). Dashed circles indicate the nuclear space; gray-shaded objects symbolize condensed chromatin. (B) FCS analysis of GFP–α-tubulin, Megator-mCherry, and mRFP-Mad2 during mitosis after MT depolymerization with colchicine. Depicted are the mean or median values of the dwell time τD and the anomaly degree (α) of the diffusional motion within the focus in both the nuclear region (N) and cytoplasm (C). ***, P < 0.001. (C–E) FCS measurements were performed at 10 loci per cell (tubulin, 11 cells; Megator, 15 cells; Mad2, 15 cells). Position data were rescaled (nuclear region: λ = −0.2, . . . , +0.2). The number of fluorescent molecules (N) within the focus was relativized to the maximum number of fluorescent molecules (Nmax) detected in each cell to account for varying expression levels. (F and G) Modeling of protein diffusion into/out of the nuclear region. Diffusion of soluble tubulin (F) into and Mad2 (G) out of the nuclear region progresses within few seconds to a steady state, as indicated by the apparent fluorescence in the nuclear region Fn(t) and the cytoplasm Fc(t). Representative fluorescence images of the initial condition, an intermediate state, and the steady state are shown in the top panels. Dashed white lines indicate the respective line scans used for quantification of fluorescence (inset graphs).
Mentions: To investigate how a fenestrated organelle-exclusion envelope drives protein accumulation in the nuclear/spindle region during early mitosis, we characterized the diffusion properties of GFP–α-tubulin and Megator-mCherry at a single-molecule level using fluorescence correlation spectroscopy (FCS) in S2 cells. To ensure the independence from MTs, they were depolymerized with colchicine. While soluble tubulin showed similar anomalous diffusion inside and outside the nuclear region, Megator diffusion was about five times slower in the nuclear region when compared with the rest of the cytoplasm (Fig. 4, A–E), where Megator diffused more anomalously (i.e., more obstructed). These differences might reflect interactions with at least two other nuclear-derived proteins: Chromator and EAST (Qi et al., 2004, 2005).

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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Related in: MedlinePlus