Limits...
Gamma-tubulin coordinates nuclear envelope assembly around chromatin

View Article: PubMed Central - PubMed

ABSTRACT

The cytosolic role of γ-tubulin as a microtubule organizer has been studied thoroughly, but its nuclear function is poorly understood. Here, we show that γ-tubulin is located throughout the chromatin of demembranated Xenopus laevis sperm and, as the nucleus is formed, γ-tubulin recruits lamin B3 and nuclear membranes. Immunodepletion of γ-tubulin impairs X. laevis assembly of both the lamina and the nuclear membrane. During nuclear formation in mammalian cell lines, γ-tubulin establishes a cellular protein boundary around chromatin that coordinates nuclear assembly of the daughter nuclei. Furthermore, expression of a γ-tubulin mutant that lacks the DNA-binding domain forms chromatin-empty nuclear like structures and demonstrate that a constant interplay between the chromatin-associated and the cytosolic pools of γ-tubulin is required and, when the balance between pools is impaired, aberrant nuclei are formed. We therefore propose that the nuclear protein meshwork formed by γ-tubulin around chromatin coordinates nuclear formation in eukaryotic cells.

No MeSH data available.


Neither RBM3 nor SadBL form a cellular meshwork. (A, B) Confocal images of a Z-stack of U2OS cells transiently expressing either GFP-RBM3 (RBM3; A) or GFP-SadBL (SadB; B), and transiently expressing mCherry-tagged lamin B (laminB1). Images were collected at 0.34 μm intervals. (A) Z-stack images taken in the intervals 0.34–0.68 μm and 2.72–3.74 μm show the nuclear envelope and in the interval 1.02–2.38 μm shows the nuclear compartment of cells expressing RBM3. (B) Z-stack images taken in the intervals 0.34–0.68 μm and 3.06–3.74 μm show the nuclear envelope and in the interval 1.02–2.72 μm shows the nuclear compartment of cells expressing SADB. (A, B) The magnified area (white border) shows the mid plane of the nuclear compartment (right panels). Scale bars, 10 μm.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037270&req=5

fig0050: Neither RBM3 nor SadBL form a cellular meshwork. (A, B) Confocal images of a Z-stack of U2OS cells transiently expressing either GFP-RBM3 (RBM3; A) or GFP-SadBL (SadB; B), and transiently expressing mCherry-tagged lamin B (laminB1). Images were collected at 0.34 μm intervals. (A) Z-stack images taken in the intervals 0.34–0.68 μm and 2.72–3.74 μm show the nuclear envelope and in the interval 1.02–2.38 μm shows the nuclear compartment of cells expressing RBM3. (B) Z-stack images taken in the intervals 0.34–0.68 μm and 3.06–3.74 μm show the nuclear envelope and in the interval 1.02–2.72 μm shows the nuclear compartment of cells expressing SADB. (A, B) The magnified area (white border) shows the mid plane of the nuclear compartment (right panels). Scale bars, 10 μm.

Mentions: To exclude the possibility that fixation of the cells caused formation of γ-tubulin strings, we studied the location of the protein centrin. In comparison with γ-tubulin, immunofluorescence staining with an anti-centrin antibody showed that centrin formed no cellular strings (Fig. 9C, D). Accordingly, the localization of both the nuclear GFP-tagged human RNA-binding protein 3 (GFP-RBM3) and of the cytosolic GFP-tagged ser/thr kinase SADB-long (mSADB, hSAD1/BRSK1; GFP-SADBL) differed from γ-tubulin in the NE. Z-stack images of whole living U2OS cells transiently expressing either GFP-RBM3 or GFP-SADBL showed that neither GFP-RBM3 nor GFP-SADBL were found to continue cross the NE (0.34–0.68 μm and 2.72–3.74 μm RBM3 and 0.34–0.68 μm and 3.06–3.74 μm SADBL; Fig. 7A, B, Fig. 9B and Fig. 10A, B). Altogether, these data confirm that γ-tubulin forms a meshwork that connects the chromatin to the cytoplasm.


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
Neither RBM3 nor SadBL form a cellular meshwork. (A, B) Confocal images of a Z-stack of U2OS cells transiently expressing either GFP-RBM3 (RBM3; A) or GFP-SadBL (SadB; B), and transiently expressing mCherry-tagged lamin B (laminB1). Images were collected at 0.34 μm intervals. (A) Z-stack images taken in the intervals 0.34–0.68 μm and 2.72–3.74 μm show the nuclear envelope and in the interval 1.02–2.38 μm shows the nuclear compartment of cells expressing RBM3. (B) Z-stack images taken in the intervals 0.34–0.68 μm and 3.06–3.74 μm show the nuclear envelope and in the interval 1.02–2.72 μm shows the nuclear compartment of cells expressing SADB. (A, B) The magnified area (white border) shows the mid plane of the nuclear compartment (right panels). Scale bars, 10 μm.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037270&req=5

fig0050: Neither RBM3 nor SadBL form a cellular meshwork. (A, B) Confocal images of a Z-stack of U2OS cells transiently expressing either GFP-RBM3 (RBM3; A) or GFP-SadBL (SadB; B), and transiently expressing mCherry-tagged lamin B (laminB1). Images were collected at 0.34 μm intervals. (A) Z-stack images taken in the intervals 0.34–0.68 μm and 2.72–3.74 μm show the nuclear envelope and in the interval 1.02–2.38 μm shows the nuclear compartment of cells expressing RBM3. (B) Z-stack images taken in the intervals 0.34–0.68 μm and 3.06–3.74 μm show the nuclear envelope and in the interval 1.02–2.72 μm shows the nuclear compartment of cells expressing SADB. (A, B) The magnified area (white border) shows the mid plane of the nuclear compartment (right panels). Scale bars, 10 μm.
Mentions: To exclude the possibility that fixation of the cells caused formation of γ-tubulin strings, we studied the location of the protein centrin. In comparison with γ-tubulin, immunofluorescence staining with an anti-centrin antibody showed that centrin formed no cellular strings (Fig. 9C, D). Accordingly, the localization of both the nuclear GFP-tagged human RNA-binding protein 3 (GFP-RBM3) and of the cytosolic GFP-tagged ser/thr kinase SADB-long (mSADB, hSAD1/BRSK1; GFP-SADBL) differed from γ-tubulin in the NE. Z-stack images of whole living U2OS cells transiently expressing either GFP-RBM3 or GFP-SADBL showed that neither GFP-RBM3 nor GFP-SADBL were found to continue cross the NE (0.34–0.68 μm and 2.72–3.74 μm RBM3 and 0.34–0.68 μm and 3.06–3.74 μm SADBL; Fig. 7A, B, Fig. 9B and Fig. 10A, B). Altogether, these data confirm that γ-tubulin forms a meshwork that connects the chromatin to the cytoplasm.

View Article: PubMed Central - PubMed

ABSTRACT

The cytosolic role of γ-tubulin as a microtubule organizer has been studied thoroughly, but its nuclear function is poorly understood. Here, we show that γ-tubulin is located throughout the chromatin of demembranated Xenopus laevis sperm and, as the nucleus is formed, γ-tubulin recruits lamin B3 and nuclear membranes. Immunodepletion of γ-tubulin impairs X. laevis assembly of both the lamina and the nuclear membrane. During nuclear formation in mammalian cell lines, γ-tubulin establishes a cellular protein boundary around chromatin that coordinates nuclear assembly of the daughter nuclei. Furthermore, expression of a γ-tubulin mutant that lacks the DNA-binding domain forms chromatin-empty nuclear like structures and demonstrate that a constant interplay between the chromatin-associated and the cytosolic pools of γ-tubulin is required and, when the balance between pools is impaired, aberrant nuclei are formed. We therefore propose that the nuclear protein meshwork formed by γ-tubulin around chromatin coordinates nuclear formation in eukaryotic cells.

No MeSH data available.