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A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing.

Umasankar PK, Ma L, Thieman JR, Jha A, Doray B, Watkins SC, Traub LM - Elife (2014)

Bottom Line: Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease.By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication.Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, United States.

ABSTRACT
Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization.

No MeSH data available.


Related in: MedlinePlus

Characterization of FCHO2- HeLa clone #64 cells.(A) Semi-quantitiative RT-PCT analysis of muniscin protein transcripts in parental HeLa SS6, clone #64 and clone#64/1.E cells. The same PCR primers as in Figure 1C were used. HC; heavy chain. (B) Whole cell lysates from HeLa SS6, clone #64 and K562 cells were resolved by SDS-PAGE and either stained with Coomassie blue or transferred to nitrocellulose. Replicate blots were probed with antibodies specific for the indicated proteins. Positions of the molecular mass standards (in kDa) are indicated on the left. (C and D). After incubation at 37°C for 60 min in serum-free medium, cover slip-attached HeLa SS6 (C) or clone #64 cells (D) were pulsed for 10 min with Alexa Fluor488-labelled transferrin (green). After chilling on ice and washing with ice-cold PBS, the cells were fixed and stained for AP-2 (red) with the α-subunit specific mAb AP.6. Representative single confocal optical sections focused on a medial region, rich in transferrin-positive endosomal structures, are shown, with color-separated channels of the boxed regions shown below. Scale bar: 10 μm.DOI:http://dx.doi.org/10.7554/eLife.04137.005
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fig3: Characterization of FCHO2- HeLa clone #64 cells.(A) Semi-quantitiative RT-PCT analysis of muniscin protein transcripts in parental HeLa SS6, clone #64 and clone#64/1.E cells. The same PCR primers as in Figure 1C were used. HC; heavy chain. (B) Whole cell lysates from HeLa SS6, clone #64 and K562 cells were resolved by SDS-PAGE and either stained with Coomassie blue or transferred to nitrocellulose. Replicate blots were probed with antibodies specific for the indicated proteins. Positions of the molecular mass standards (in kDa) are indicated on the left. (C and D). After incubation at 37°C for 60 min in serum-free medium, cover slip-attached HeLa SS6 (C) or clone #64 cells (D) were pulsed for 10 min with Alexa Fluor488-labelled transferrin (green). After chilling on ice and washing with ice-cold PBS, the cells were fixed and stained for AP-2 (red) with the α-subunit specific mAb AP.6. Representative single confocal optical sections focused on a medial region, rich in transferrin-positive endosomal structures, are shown, with color-separated channels of the boxed regions shown below. Scale bar: 10 μm.DOI:http://dx.doi.org/10.7554/eLife.04137.005

Mentions: We selected clone #64 for further detailed analysis as this cell has two distinct 4-bp deletions, each leading to an immediate in-frame stop codon (Q97X) terminating the FCHO1 polypeptide within the EFC domain (Figure 1E). RT-PCR shows a strong qualitative decrease in FCHO2 mRNA abundance in clone #64 cells (Figure 3A), presumably due to nonsense-mediated decay. In these FCHO2- cells, the relative levels of numerous other endocytic and some unrelated cellular proteins are essentially unaltered (Figure 3B). Notably, FCHO1, undetectable by immunoblotting in HeLa cells (Uezu et al., 2011; Umasankar et al., 2012; Figure 3B) does not show a compensatory increase in clone #64 cells, either at the RNA or protein level, clearly dissimilar to the counteracting expression reported in BS-C-1 cells (Nunez et al., 2011). Endogenous FCHO1 is readily detectable, along with FCHO2, in K562 erythroleukemia cells. RNA-seq results indicate an eightfold higher FCHO1 expression in K562 cells compared with HeLa (Lundberg et al., 2010; Uhlen et al., 2010). Moreover, RT-PCR fails to detect evidence of the SGIP1 transcript in either HeLa or clone #64 cells (Figure 3A). Indeed, SGIP1 is essentially a neuronally-expressed protein (Trevaskis et al., 2005; Uezu et al., 2007), with RT-PCR (Figure 1C) and RNA-seq (Lundberg et al., 2010; Uhlen et al., 2010) indicating >30-fold higher transcript abundance in SH-SY5Y neuroblastoma cells vs HeLa cells.10.7554/eLife.04137.005Figure 3.Characterization of FCHO2- HeLa clone #64 cells.


A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing.

Umasankar PK, Ma L, Thieman JR, Jha A, Doray B, Watkins SC, Traub LM - Elife (2014)

Characterization of FCHO2- HeLa clone #64 cells.(A) Semi-quantitiative RT-PCT analysis of muniscin protein transcripts in parental HeLa SS6, clone #64 and clone#64/1.E cells. The same PCR primers as in Figure 1C were used. HC; heavy chain. (B) Whole cell lysates from HeLa SS6, clone #64 and K562 cells were resolved by SDS-PAGE and either stained with Coomassie blue or transferred to nitrocellulose. Replicate blots were probed with antibodies specific for the indicated proteins. Positions of the molecular mass standards (in kDa) are indicated on the left. (C and D). After incubation at 37°C for 60 min in serum-free medium, cover slip-attached HeLa SS6 (C) or clone #64 cells (D) were pulsed for 10 min with Alexa Fluor488-labelled transferrin (green). After chilling on ice and washing with ice-cold PBS, the cells were fixed and stained for AP-2 (red) with the α-subunit specific mAb AP.6. Representative single confocal optical sections focused on a medial region, rich in transferrin-positive endosomal structures, are shown, with color-separated channels of the boxed regions shown below. Scale bar: 10 μm.DOI:http://dx.doi.org/10.7554/eLife.04137.005
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215538&req=5

fig3: Characterization of FCHO2- HeLa clone #64 cells.(A) Semi-quantitiative RT-PCT analysis of muniscin protein transcripts in parental HeLa SS6, clone #64 and clone#64/1.E cells. The same PCR primers as in Figure 1C were used. HC; heavy chain. (B) Whole cell lysates from HeLa SS6, clone #64 and K562 cells were resolved by SDS-PAGE and either stained with Coomassie blue or transferred to nitrocellulose. Replicate blots were probed with antibodies specific for the indicated proteins. Positions of the molecular mass standards (in kDa) are indicated on the left. (C and D). After incubation at 37°C for 60 min in serum-free medium, cover slip-attached HeLa SS6 (C) or clone #64 cells (D) were pulsed for 10 min with Alexa Fluor488-labelled transferrin (green). After chilling on ice and washing with ice-cold PBS, the cells were fixed and stained for AP-2 (red) with the α-subunit specific mAb AP.6. Representative single confocal optical sections focused on a medial region, rich in transferrin-positive endosomal structures, are shown, with color-separated channels of the boxed regions shown below. Scale bar: 10 μm.DOI:http://dx.doi.org/10.7554/eLife.04137.005
Mentions: We selected clone #64 for further detailed analysis as this cell has two distinct 4-bp deletions, each leading to an immediate in-frame stop codon (Q97X) terminating the FCHO1 polypeptide within the EFC domain (Figure 1E). RT-PCR shows a strong qualitative decrease in FCHO2 mRNA abundance in clone #64 cells (Figure 3A), presumably due to nonsense-mediated decay. In these FCHO2- cells, the relative levels of numerous other endocytic and some unrelated cellular proteins are essentially unaltered (Figure 3B). Notably, FCHO1, undetectable by immunoblotting in HeLa cells (Uezu et al., 2011; Umasankar et al., 2012; Figure 3B) does not show a compensatory increase in clone #64 cells, either at the RNA or protein level, clearly dissimilar to the counteracting expression reported in BS-C-1 cells (Nunez et al., 2011). Endogenous FCHO1 is readily detectable, along with FCHO2, in K562 erythroleukemia cells. RNA-seq results indicate an eightfold higher FCHO1 expression in K562 cells compared with HeLa (Lundberg et al., 2010; Uhlen et al., 2010). Moreover, RT-PCR fails to detect evidence of the SGIP1 transcript in either HeLa or clone #64 cells (Figure 3A). Indeed, SGIP1 is essentially a neuronally-expressed protein (Trevaskis et al., 2005; Uezu et al., 2007), with RT-PCR (Figure 1C) and RNA-seq (Lundberg et al., 2010; Uhlen et al., 2010) indicating >30-fold higher transcript abundance in SH-SY5Y neuroblastoma cells vs HeLa cells.10.7554/eLife.04137.005Figure 3.Characterization of FCHO2- HeLa clone #64 cells.

Bottom Line: Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease.By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication.Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, United States.

ABSTRACT
Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization.

No MeSH data available.


Related in: MedlinePlus