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Syntaxin 16 is a master recruitment factor for cytokinesis.

Neto H, Kaupisch A, Collins LL, Gould GW - Mol. Biol. Cell (2013)

Bottom Line: However, it is not clear how either of these complexes is targeted to the midbody and whether their delivery is coordinated.Although membrane traffic is known to play an important role in cytokinesis, the contribution and identity of intracellular SNAREs to cytokinesis remain unclear.Here we demonstrate that syntaxin 16 is a key regulator of cytokinesis, as it is required for recruitment of both recycling endosome-associated Exocyst and ESCRT machinery during late telophase, and therefore that these two distinct facets of cytokinesis are inextricably linked.

View Article: PubMed Central - PubMed

Affiliation: Henry Wellcome Laboratory of Cell Biology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom.

ABSTRACT
Recently it was shown that both recycling endosome and endosomal sorting complex required for transport (ESCRT) components are required for cytokinesis, in which they are believed to act in a sequential manner to bring about secondary ingression and abscission, respectively. However, it is not clear how either of these complexes is targeted to the midbody and whether their delivery is coordinated. The trafficking of membrane vesicles between different intracellular organelles involves the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. Although membrane traffic is known to play an important role in cytokinesis, the contribution and identity of intracellular SNAREs to cytokinesis remain unclear. Here we demonstrate that syntaxin 16 is a key regulator of cytokinesis, as it is required for recruitment of both recycling endosome-associated Exocyst and ESCRT machinery during late telophase, and therefore that these two distinct facets of cytokinesis are inextricably linked.

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Syntaxin 16 is required for cytokinesis. HeLa cells were incubated with adenovirus designed to express the indicated Sx-ΔTM construct at identical multiplicity of infection (in the experiment shown, 30:1), empty virus or not infected (control), as described in Materials and Methods. At 48 h later, cells were fixed and stained with anti-tubulin antibodies and DAPI and the frequency of binucleate cells counted. (A) Quantification of five independent experiments of this type. Sx6-ΔTM and Sx16-ΔTM significantly increased the frequency of binucleate cells (*p < 0.05), using at least three different batches of virus. (B) Typical fields of cells infected with Sx12-ΔTM or Sx16-ΔTM as indicated and stained for tubulin (red; tubulin), Sx16 (green; Sx16) or DNA (blue; DAPI). Note that for Sx16-ΔTM–infected cells, the high level of overexpression of Sx16-ΔTM required the use of reduced gain/laser power during collection of the image. (C) Comparison of the frequency of binucleate cells in cells infected with empty virus, Sx16-ΔTM, or Sx16-full length. (D) HeLa cells were transfected with an siRNA SmartPool designed to knock down Sx16 or a scrambled siRNA SmartPool control and then incubated for 48 h after transfection before fixation and staining with anti-Sx16. Data from a typical experiment. Left, typical cell in telophase, with endogenous Sx16 present near the midbody. See Figure 3 for Sx16 protein levels after knockdown. C and D are representative of three or more experiments of this type.
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Figure 1: Syntaxin 16 is required for cytokinesis. HeLa cells were incubated with adenovirus designed to express the indicated Sx-ΔTM construct at identical multiplicity of infection (in the experiment shown, 30:1), empty virus or not infected (control), as described in Materials and Methods. At 48 h later, cells were fixed and stained with anti-tubulin antibodies and DAPI and the frequency of binucleate cells counted. (A) Quantification of five independent experiments of this type. Sx6-ΔTM and Sx16-ΔTM significantly increased the frequency of binucleate cells (*p < 0.05), using at least three different batches of virus. (B) Typical fields of cells infected with Sx12-ΔTM or Sx16-ΔTM as indicated and stained for tubulin (red; tubulin), Sx16 (green; Sx16) or DNA (blue; DAPI). Note that for Sx16-ΔTM–infected cells, the high level of overexpression of Sx16-ΔTM required the use of reduced gain/laser power during collection of the image. (C) Comparison of the frequency of binucleate cells in cells infected with empty virus, Sx16-ΔTM, or Sx16-full length. (D) HeLa cells were transfected with an siRNA SmartPool designed to knock down Sx16 or a scrambled siRNA SmartPool control and then incubated for 48 h after transfection before fixation and staining with anti-Sx16. Data from a typical experiment. Left, typical cell in telophase, with endogenous Sx16 present near the midbody. See Figure 3 for Sx16 protein levels after knockdown. C and D are representative of three or more experiments of this type.

Mentions: In an effort to determine which endosomal t-SNAREs may be involved in membrane trafficking in cytokinesis, we assayed the effect on cytokinesis of adenoviral-driven overexpression of t-SNAREs devoid of their transmembrane domain (hereafter referred to as -ΔTM mutants). Such mutants were previously shown to interfere with the endogenous t-SNAREs by acting as dominant interfering mutants (see, e.g., Scales et al., 2000; Perera et al., 2003; Choudhury et al., 2006; Proctor et al., 2006). HeLa cells were infected with recombinant adenovirus engineered to overexpress Sx6-ΔTM, Sx8-ΔTM, Sx12-ΔTM, or Sx16-ΔTM (see Supplemental Figure S1A) and the consequences for cytokinesis determined by quantifying the frequency of binuclear cells and comparing this with control (uninfected) cells or cells infected with empty adenovirus. Note that under the conditions used in all experiments reported here (multiplicity of infection of 30:1), >98% of cells express the mutant Sx-ΔTM in question (Supplemental Figure S1B). Figure 1, A and B, shows that overexpression of Sx6-ΔTM or Sx16-ΔTM, but not Sx8-ΔTM or Sx12-ΔTM, significantly increased the frequency of binucleate cells, suggestive of defective cytokinesis. The observed increase in binuclear cells was not observed upon overexpression of wild-type (full-length) Sx16 (Figure 1C). A potential role for Sx16 in cytokinesis is supported by our observation that endogenous Sx16 localized to the midbody in late telophase (Figure 1D); note that the specificity of antibody staining was verified by comparing signals in cells depleted of Sx16 by siRNA (Figure 1D). Finally, real-time image analysis revealed that overexpression of Sx16-ΔTM consistently increased the time taken for completion of abscission, such that 100% of cells examined (>25) took >2 h to proceed from the onset of furrowing to completion of abscission and ∼35% became binucleate; by contrast, >80% of control cells completed abscission in a time of <2 h, and none were binucleate (Figure 2). Knockdown of Sx6 or Sx16 using siRNA also resulted in a marked increase in the frequency of binuclear cells (Figure 3). Such data collectively argue that Sx6 and Sx16 play an important role in cytokinesis. Sx16 is a Qa-SNARE, known to interact with the Sec1/Munc18 family member mVps45 (Tellam et al., 1997; Simonsen et al., 1998; Dulubova et al., 2002; Struthers et al., 2009). Given that Sec1/Munc18 proteins are known to act as regulators of the SNARE machinery, we tested whether mVps45 is also required for cytokinesis. Knockdown of mVps45 by siRNA also resulted in a dramatic increase in the number of binucleate cells (Figure 2).


Syntaxin 16 is a master recruitment factor for cytokinesis.

Neto H, Kaupisch A, Collins LL, Gould GW - Mol. Biol. Cell (2013)

Syntaxin 16 is required for cytokinesis. HeLa cells were incubated with adenovirus designed to express the indicated Sx-ΔTM construct at identical multiplicity of infection (in the experiment shown, 30:1), empty virus or not infected (control), as described in Materials and Methods. At 48 h later, cells were fixed and stained with anti-tubulin antibodies and DAPI and the frequency of binucleate cells counted. (A) Quantification of five independent experiments of this type. Sx6-ΔTM and Sx16-ΔTM significantly increased the frequency of binucleate cells (*p < 0.05), using at least three different batches of virus. (B) Typical fields of cells infected with Sx12-ΔTM or Sx16-ΔTM as indicated and stained for tubulin (red; tubulin), Sx16 (green; Sx16) or DNA (blue; DAPI). Note that for Sx16-ΔTM–infected cells, the high level of overexpression of Sx16-ΔTM required the use of reduced gain/laser power during collection of the image. (C) Comparison of the frequency of binucleate cells in cells infected with empty virus, Sx16-ΔTM, or Sx16-full length. (D) HeLa cells were transfected with an siRNA SmartPool designed to knock down Sx16 or a scrambled siRNA SmartPool control and then incubated for 48 h after transfection before fixation and staining with anti-Sx16. Data from a typical experiment. Left, typical cell in telophase, with endogenous Sx16 present near the midbody. See Figure 3 for Sx16 protein levels after knockdown. C and D are representative of three or more experiments of this type.
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Figure 1: Syntaxin 16 is required for cytokinesis. HeLa cells were incubated with adenovirus designed to express the indicated Sx-ΔTM construct at identical multiplicity of infection (in the experiment shown, 30:1), empty virus or not infected (control), as described in Materials and Methods. At 48 h later, cells were fixed and stained with anti-tubulin antibodies and DAPI and the frequency of binucleate cells counted. (A) Quantification of five independent experiments of this type. Sx6-ΔTM and Sx16-ΔTM significantly increased the frequency of binucleate cells (*p < 0.05), using at least three different batches of virus. (B) Typical fields of cells infected with Sx12-ΔTM or Sx16-ΔTM as indicated and stained for tubulin (red; tubulin), Sx16 (green; Sx16) or DNA (blue; DAPI). Note that for Sx16-ΔTM–infected cells, the high level of overexpression of Sx16-ΔTM required the use of reduced gain/laser power during collection of the image. (C) Comparison of the frequency of binucleate cells in cells infected with empty virus, Sx16-ΔTM, or Sx16-full length. (D) HeLa cells were transfected with an siRNA SmartPool designed to knock down Sx16 or a scrambled siRNA SmartPool control and then incubated for 48 h after transfection before fixation and staining with anti-Sx16. Data from a typical experiment. Left, typical cell in telophase, with endogenous Sx16 present near the midbody. See Figure 3 for Sx16 protein levels after knockdown. C and D are representative of three or more experiments of this type.
Mentions: In an effort to determine which endosomal t-SNAREs may be involved in membrane trafficking in cytokinesis, we assayed the effect on cytokinesis of adenoviral-driven overexpression of t-SNAREs devoid of their transmembrane domain (hereafter referred to as -ΔTM mutants). Such mutants were previously shown to interfere with the endogenous t-SNAREs by acting as dominant interfering mutants (see, e.g., Scales et al., 2000; Perera et al., 2003; Choudhury et al., 2006; Proctor et al., 2006). HeLa cells were infected with recombinant adenovirus engineered to overexpress Sx6-ΔTM, Sx8-ΔTM, Sx12-ΔTM, or Sx16-ΔTM (see Supplemental Figure S1A) and the consequences for cytokinesis determined by quantifying the frequency of binuclear cells and comparing this with control (uninfected) cells or cells infected with empty adenovirus. Note that under the conditions used in all experiments reported here (multiplicity of infection of 30:1), >98% of cells express the mutant Sx-ΔTM in question (Supplemental Figure S1B). Figure 1, A and B, shows that overexpression of Sx6-ΔTM or Sx16-ΔTM, but not Sx8-ΔTM or Sx12-ΔTM, significantly increased the frequency of binucleate cells, suggestive of defective cytokinesis. The observed increase in binuclear cells was not observed upon overexpression of wild-type (full-length) Sx16 (Figure 1C). A potential role for Sx16 in cytokinesis is supported by our observation that endogenous Sx16 localized to the midbody in late telophase (Figure 1D); note that the specificity of antibody staining was verified by comparing signals in cells depleted of Sx16 by siRNA (Figure 1D). Finally, real-time image analysis revealed that overexpression of Sx16-ΔTM consistently increased the time taken for completion of abscission, such that 100% of cells examined (>25) took >2 h to proceed from the onset of furrowing to completion of abscission and ∼35% became binucleate; by contrast, >80% of control cells completed abscission in a time of <2 h, and none were binucleate (Figure 2). Knockdown of Sx6 or Sx16 using siRNA also resulted in a marked increase in the frequency of binuclear cells (Figure 3). Such data collectively argue that Sx6 and Sx16 play an important role in cytokinesis. Sx16 is a Qa-SNARE, known to interact with the Sec1/Munc18 family member mVps45 (Tellam et al., 1997; Simonsen et al., 1998; Dulubova et al., 2002; Struthers et al., 2009). Given that Sec1/Munc18 proteins are known to act as regulators of the SNARE machinery, we tested whether mVps45 is also required for cytokinesis. Knockdown of mVps45 by siRNA also resulted in a dramatic increase in the number of binucleate cells (Figure 2).

Bottom Line: However, it is not clear how either of these complexes is targeted to the midbody and whether their delivery is coordinated.Although membrane traffic is known to play an important role in cytokinesis, the contribution and identity of intracellular SNAREs to cytokinesis remain unclear.Here we demonstrate that syntaxin 16 is a key regulator of cytokinesis, as it is required for recruitment of both recycling endosome-associated Exocyst and ESCRT machinery during late telophase, and therefore that these two distinct facets of cytokinesis are inextricably linked.

View Article: PubMed Central - PubMed

Affiliation: Henry Wellcome Laboratory of Cell Biology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom.

ABSTRACT
Recently it was shown that both recycling endosome and endosomal sorting complex required for transport (ESCRT) components are required for cytokinesis, in which they are believed to act in a sequential manner to bring about secondary ingression and abscission, respectively. However, it is not clear how either of these complexes is targeted to the midbody and whether their delivery is coordinated. The trafficking of membrane vesicles between different intracellular organelles involves the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. Although membrane traffic is known to play an important role in cytokinesis, the contribution and identity of intracellular SNAREs to cytokinesis remain unclear. Here we demonstrate that syntaxin 16 is a key regulator of cytokinesis, as it is required for recruitment of both recycling endosome-associated Exocyst and ESCRT machinery during late telophase, and therefore that these two distinct facets of cytokinesis are inextricably linked.

Show MeSH
Related in: MedlinePlus