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Mechanism of Ca²⁺-triggered ESCRT assembly and regulation of cell membrane repair.

Scheffer LL, Sreetama SC, Sharma N, Medikayala S, Brown KJ, Defour A, Jaiswal JK - Nat Commun (2014)

Bottom Line: Here, we show that injury-triggered Ca(2+) increase results in assembly of ESCRT III and accessory proteins at the site of repair.This process is initiated by the calcium-binding protein-apoptosis-linked gene (ALG)-2.ALG-2 facilitates accumulation of ALG-2-interacting protein X (ALIX), ESCRT III and Vps4 complex at the injured cell membrane, which in turn results in cleavage and shedding of the damaged part of the cell membrane.

View Article: PubMed Central - PubMed

Affiliation: Children's National Medical Center, Center for Genetic Medicine Research, 111 Michigan Avenue, NW, Washington DC 20010-2970, USA.

ABSTRACT
In muscle and other mechanically active tissue, cell membranes are constantly injured, and their repair depends on the injury-induced increase in cytosolic calcium. Here, we show that injury-triggered Ca(2+) increase results in assembly of ESCRT III and accessory proteins at the site of repair. This process is initiated by the calcium-binding protein-apoptosis-linked gene (ALG)-2. ALG-2 facilitates accumulation of ALG-2-interacting protein X (ALIX), ESCRT III and Vps4 complex at the injured cell membrane, which in turn results in cleavage and shedding of the damaged part of the cell membrane. Lack of ALG-2, ALIX or Vps4B each prevents shedding, and repair of the injured cell membrane. These results demonstrate Ca(2+)-dependent accumulation of ESCRT III-Vps4 complex following large focal injury to the cell membrane and identify the role of ALG-2 as the initiator of sequential ESCRT III-Vps4 complex assembly that facilitates scission and repair of the injured cell membrane.

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ALG-2 is required for injury-triggered ALIX and ESCRT III assemblyControl and ALG-2 specific siRNAs were transfected in HeLa cells and its effect was assessed on (A) expression of ALG-2 and ALIX (see Supplementary Fig. 5A for uncropped blots), (B-G) injury-triggered accumulation of (B, C) Vps4B-GFP, (D, E) Chmp4B-YFP, and (F, G) ALIX-GFP. TIRF images show cells 1s prior to injury (Pre-injury) and 125s following injury (post-injury). Plots show change in intensity of indicated protein at the site of repair in 4 cells each. The site of injury is marked by white arrow while the site of repair is marked by the dotted box. Scale bar represents 10μm and the error bars indicate SE.
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Figure 4: ALG-2 is required for injury-triggered ALIX and ESCRT III assemblyControl and ALG-2 specific siRNAs were transfected in HeLa cells and its effect was assessed on (A) expression of ALG-2 and ALIX (see Supplementary Fig. 5A for uncropped blots), (B-G) injury-triggered accumulation of (B, C) Vps4B-GFP, (D, E) Chmp4B-YFP, and (F, G) ALIX-GFP. TIRF images show cells 1s prior to injury (Pre-injury) and 125s following injury (post-injury). Plots show change in intensity of indicated protein at the site of repair in 4 cells each. The site of injury is marked by white arrow while the site of repair is marked by the dotted box. Scale bar represents 10μm and the error bars indicate SE.

Mentions: An important feature of ESCRT III accumulation at the injured cell membrane is its Ca2+-dependence. This could be on account of the EF-hand containing Ca2+-binding protein ALG-2 that has been shown to associate with ALIX, TSG-101, and Chmp4B in a Ca2+-dependent manner30. To examine if ALG-2 regulates Ca2+-triggered accumulation of ESCRTs we tested if lack of ALG-2 affects injury-triggered accumulation of ESCRT III and accessory proteins. By use of ALG-2 targeted siRNA in HeLa cells we reduced ALG-2 expression to well below 10% of the endogenous level (Fig.4A). Unlike the control siRNA transfected cells, the cells lacking ALG-2 expression failed to accumulate Vps4B (Figure 4B, C) and Chmp4B (Fig 4D, E) following injury. In view of the co-accumulation of ALG-2 with ALIX we tested if ALG-2 regulates ALIX accumulation or if these two proteins accumulate independently. Again, while the control siRNA had no effect on ALIX accumulation, lack of ALG-2 prevented injury-triggered accumulation of ALIX (Fig. 4F, G). While this indicates requirement of ALG-2 for ALIX accumulation, it is plausible that ALG-2 and ALIX may have complimentary role in their accumulation. In such an event ALG-2 will have a regulatory function rather than a recruitment function and each of these proteins will be needed for the co-accumulation. To test this possibility next we generated a pool of HeLa cells stably expressing ALIX shRNA vector or the control empty shRNA vector. Cells with ALIX shRNA showed <10% of ALIX protein level as compared to the empty vector control cells (Fig. 5A,B). In view of the direct interaction between ALG-2 and ALIX we tested the level of ALG-2 in the ALIX knocked down cells and found ALIX knockdown does not effect expression level of ALG-2 protein (Fig. 5A,C). We then assessed the effect of ALIX knockdown on injury-triggered ALG-2 accumulation and found that lack of ALIX did not prevent, but caused a slight delay in the accumulation of ALG-2 (Fig. 5D,E). Further, similar to the lack of ALG-2, lack of ALIX also prevented injury-triggered accumulation of Chmp4B (Fig. 5F-G) and Vps4B (Fig. 5H-I). Above observations that ALG-2 and ALIX are required for injury-triggered recruitment of ESCRT III and Vps4 complex and following injury they begin accumulating prior to ESCRT III and Vps4 (Fig. 2N) point to sequential recruitment of injury-triggered ESCRT accumulation, which is initiated by ALG-2 and mediated by ALIX.


Mechanism of Ca²⁺-triggered ESCRT assembly and regulation of cell membrane repair.

Scheffer LL, Sreetama SC, Sharma N, Medikayala S, Brown KJ, Defour A, Jaiswal JK - Nat Commun (2014)

ALG-2 is required for injury-triggered ALIX and ESCRT III assemblyControl and ALG-2 specific siRNAs were transfected in HeLa cells and its effect was assessed on (A) expression of ALG-2 and ALIX (see Supplementary Fig. 5A for uncropped blots), (B-G) injury-triggered accumulation of (B, C) Vps4B-GFP, (D, E) Chmp4B-YFP, and (F, G) ALIX-GFP. TIRF images show cells 1s prior to injury (Pre-injury) and 125s following injury (post-injury). Plots show change in intensity of indicated protein at the site of repair in 4 cells each. The site of injury is marked by white arrow while the site of repair is marked by the dotted box. Scale bar represents 10μm and the error bars indicate SE.
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Related In: Results  -  Collection

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Figure 4: ALG-2 is required for injury-triggered ALIX and ESCRT III assemblyControl and ALG-2 specific siRNAs were transfected in HeLa cells and its effect was assessed on (A) expression of ALG-2 and ALIX (see Supplementary Fig. 5A for uncropped blots), (B-G) injury-triggered accumulation of (B, C) Vps4B-GFP, (D, E) Chmp4B-YFP, and (F, G) ALIX-GFP. TIRF images show cells 1s prior to injury (Pre-injury) and 125s following injury (post-injury). Plots show change in intensity of indicated protein at the site of repair in 4 cells each. The site of injury is marked by white arrow while the site of repair is marked by the dotted box. Scale bar represents 10μm and the error bars indicate SE.
Mentions: An important feature of ESCRT III accumulation at the injured cell membrane is its Ca2+-dependence. This could be on account of the EF-hand containing Ca2+-binding protein ALG-2 that has been shown to associate with ALIX, TSG-101, and Chmp4B in a Ca2+-dependent manner30. To examine if ALG-2 regulates Ca2+-triggered accumulation of ESCRTs we tested if lack of ALG-2 affects injury-triggered accumulation of ESCRT III and accessory proteins. By use of ALG-2 targeted siRNA in HeLa cells we reduced ALG-2 expression to well below 10% of the endogenous level (Fig.4A). Unlike the control siRNA transfected cells, the cells lacking ALG-2 expression failed to accumulate Vps4B (Figure 4B, C) and Chmp4B (Fig 4D, E) following injury. In view of the co-accumulation of ALG-2 with ALIX we tested if ALG-2 regulates ALIX accumulation or if these two proteins accumulate independently. Again, while the control siRNA had no effect on ALIX accumulation, lack of ALG-2 prevented injury-triggered accumulation of ALIX (Fig. 4F, G). While this indicates requirement of ALG-2 for ALIX accumulation, it is plausible that ALG-2 and ALIX may have complimentary role in their accumulation. In such an event ALG-2 will have a regulatory function rather than a recruitment function and each of these proteins will be needed for the co-accumulation. To test this possibility next we generated a pool of HeLa cells stably expressing ALIX shRNA vector or the control empty shRNA vector. Cells with ALIX shRNA showed <10% of ALIX protein level as compared to the empty vector control cells (Fig. 5A,B). In view of the direct interaction between ALG-2 and ALIX we tested the level of ALG-2 in the ALIX knocked down cells and found ALIX knockdown does not effect expression level of ALG-2 protein (Fig. 5A,C). We then assessed the effect of ALIX knockdown on injury-triggered ALG-2 accumulation and found that lack of ALIX did not prevent, but caused a slight delay in the accumulation of ALG-2 (Fig. 5D,E). Further, similar to the lack of ALG-2, lack of ALIX also prevented injury-triggered accumulation of Chmp4B (Fig. 5F-G) and Vps4B (Fig. 5H-I). Above observations that ALG-2 and ALIX are required for injury-triggered recruitment of ESCRT III and Vps4 complex and following injury they begin accumulating prior to ESCRT III and Vps4 (Fig. 2N) point to sequential recruitment of injury-triggered ESCRT accumulation, which is initiated by ALG-2 and mediated by ALIX.

Bottom Line: Here, we show that injury-triggered Ca(2+) increase results in assembly of ESCRT III and accessory proteins at the site of repair.This process is initiated by the calcium-binding protein-apoptosis-linked gene (ALG)-2.ALG-2 facilitates accumulation of ALG-2-interacting protein X (ALIX), ESCRT III and Vps4 complex at the injured cell membrane, which in turn results in cleavage and shedding of the damaged part of the cell membrane.

View Article: PubMed Central - PubMed

Affiliation: Children's National Medical Center, Center for Genetic Medicine Research, 111 Michigan Avenue, NW, Washington DC 20010-2970, USA.

ABSTRACT
In muscle and other mechanically active tissue, cell membranes are constantly injured, and their repair depends on the injury-induced increase in cytosolic calcium. Here, we show that injury-triggered Ca(2+) increase results in assembly of ESCRT III and accessory proteins at the site of repair. This process is initiated by the calcium-binding protein-apoptosis-linked gene (ALG)-2. ALG-2 facilitates accumulation of ALG-2-interacting protein X (ALIX), ESCRT III and Vps4 complex at the injured cell membrane, which in turn results in cleavage and shedding of the damaged part of the cell membrane. Lack of ALG-2, ALIX or Vps4B each prevents shedding, and repair of the injured cell membrane. These results demonstrate Ca(2+)-dependent accumulation of ESCRT III-Vps4 complex following large focal injury to the cell membrane and identify the role of ALG-2 as the initiator of sequential ESCRT III-Vps4 complex assembly that facilitates scission and repair of the injured cell membrane.

Show MeSH
Related in: MedlinePlus