Limits...
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

Gene editing the FCHO2 locus in HeLa cells.(A) Domain arrangement of Homo sapiens (Hs) and S. cerevisiae (Sc) muniscin family proteins. The crescent-shaped EFC domain (Extended FCH (Fer/Cip4 homology) domain) is alternatively designated the F-BAR domain due to an overall structural homology of the α-helical anti-parallel EFC dimer to the BAR (Bin1/amphiphisin/Rvs) domain family of proteins. (B) Chromosomal location and genomic organization of the FCHO2 gene with pertinent details of TALEN design. The repeat variable di-residues (RVD) selective for the different deoxyribonucleotides are color-coded (single letter amino acid notation). The endogenous AseI recognition sequence within the targeted exon is boxed (yellow). (C) Gene-specific RT-PCR analysis of various endocytic protein and control mRNA transcripts in the parental HeLa SS6 and neuroblastoma SH-SY5Y cells. HC; heavy chain. (D) AseI restriction enzyme digestion of FCHO2 gene-specific PCR amplicons from genomic DNA extracted from wild-type (WT) and TALEN-treated clones. The undigested parental (HeLa) PCR product and digested PCRs are shown. The ‘pool’ designates a PCR reaction from a genomic DNA sample of TALEN-transfetced HeLa cells prior to clone selection. The AseI nuclease generates three PCR DNA fragments; the 55-bp band is not visible on these gels but causes the shift in the singly-cleaved product to 645 bp. (E) Genomic sequence analysis of TALEN clones. TALEN generated insertions (lower case letters) and deletions are indicated in relation to the WT nucleotide and amino acid sequences. AseI restriction sites are boxed (yellow) and in-frame stop codons are highlighted (red) and identified with a red asterisk.DOI:http://dx.doi.org/10.7554/eLife.04137.003
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215538&req=5

fig1: Gene editing the FCHO2 locus in HeLa cells.(A) Domain arrangement of Homo sapiens (Hs) and S. cerevisiae (Sc) muniscin family proteins. The crescent-shaped EFC domain (Extended FCH (Fer/Cip4 homology) domain) is alternatively designated the F-BAR domain due to an overall structural homology of the α-helical anti-parallel EFC dimer to the BAR (Bin1/amphiphisin/Rvs) domain family of proteins. (B) Chromosomal location and genomic organization of the FCHO2 gene with pertinent details of TALEN design. The repeat variable di-residues (RVD) selective for the different deoxyribonucleotides are color-coded (single letter amino acid notation). The endogenous AseI recognition sequence within the targeted exon is boxed (yellow). (C) Gene-specific RT-PCR analysis of various endocytic protein and control mRNA transcripts in the parental HeLa SS6 and neuroblastoma SH-SY5Y cells. HC; heavy chain. (D) AseI restriction enzyme digestion of FCHO2 gene-specific PCR amplicons from genomic DNA extracted from wild-type (WT) and TALEN-treated clones. The undigested parental (HeLa) PCR product and digested PCRs are shown. The ‘pool’ designates a PCR reaction from a genomic DNA sample of TALEN-transfetced HeLa cells prior to clone selection. The AseI nuclease generates three PCR DNA fragments; the 55-bp band is not visible on these gels but causes the shift in the singly-cleaved product to 645 bp. (E) Genomic sequence analysis of TALEN clones. TALEN generated insertions (lower case letters) and deletions are indicated in relation to the WT nucleotide and amino acid sequences. AseI restriction sites are boxed (yellow) and in-frame stop codons are highlighted (red) and identified with a red asterisk.DOI:http://dx.doi.org/10.7554/eLife.04137.003

Mentions: FCHO1 (KIAA0290; located on chromosome 19) and FCHO2 (on chromosome 5) display ∼50% overall amino acid identity and a similar domain organization (Figure 1A; Katoh, 2004). Both are EFC domain proteins (Shimada et al., 2007) characterized by a C-terminal μ-homology domain (μHD), distantly related in primary sequence but with an analogous fold to the cargo-engaging μ2 subunit of the heterotetrameric AP-2 clathrin adaptor (Reider et al., 2009). This combination of an EFC domain (alternatively designated the F-BAR domain [Frost et al., 2008; Heath and Insall, 2008]) with a μHD is unique, and these paralogous proteins have orthologues in chordates, arthropods and nematodes (Katoh, 2004; Umasankar et al., 2012; Mayers et al., 2013), as well as in unicellular eukaryotes (Boettner et al., 2009; Reider et al., 2009; Stimpson et al., 2009). In the budding yeast Saccharomyces cerevisiae, Syp1p has an analogous architecture to FCHO1/2 despite limited (<20%) overall primary sequence identity (Figure 1A). Phylogenetic dendrograms (TreeFam TF328986) (Ruan et al., 2008) show that a third chordate member, Sgip1, is more closely related to FCHO2 than to FCHO1, but this neuronal protein, while containing a modular C-terminal μHD, lacks the folded N-terminal helical EFC domain (Figure 1A; Uezu et al., 2007; Dergai et al., 2010). At steady state, endogenous FCHO2 (Henne et al., 2010; Umasankar et al., 2012) and both transfected FCHO1 (Reider et al., 2009; Taylor et al., 2011; Umasankar et al., 2012) and Sgip1 (Uezu et al., 2007; Stimpson et al., 2009) colocalize extensively with clathrin-coated structures, unlike several other EFC domain proteins (Cip4/Fbp17/Pacsin) implicated in endocytosis (Modregger et al., 2000; Kamioka et al., 2004; Taylor et al., 2011).10.7554/eLife.04137.003Figure 1.Gene editing the FCHO2 locus in HeLa 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)

Gene editing the FCHO2 locus in HeLa cells.(A) Domain arrangement of Homo sapiens (Hs) and S. cerevisiae (Sc) muniscin family proteins. The crescent-shaped EFC domain (Extended FCH (Fer/Cip4 homology) domain) is alternatively designated the F-BAR domain due to an overall structural homology of the α-helical anti-parallel EFC dimer to the BAR (Bin1/amphiphisin/Rvs) domain family of proteins. (B) Chromosomal location and genomic organization of the FCHO2 gene with pertinent details of TALEN design. The repeat variable di-residues (RVD) selective for the different deoxyribonucleotides are color-coded (single letter amino acid notation). The endogenous AseI recognition sequence within the targeted exon is boxed (yellow). (C) Gene-specific RT-PCR analysis of various endocytic protein and control mRNA transcripts in the parental HeLa SS6 and neuroblastoma SH-SY5Y cells. HC; heavy chain. (D) AseI restriction enzyme digestion of FCHO2 gene-specific PCR amplicons from genomic DNA extracted from wild-type (WT) and TALEN-treated clones. The undigested parental (HeLa) PCR product and digested PCRs are shown. The ‘pool’ designates a PCR reaction from a genomic DNA sample of TALEN-transfetced HeLa cells prior to clone selection. The AseI nuclease generates three PCR DNA fragments; the 55-bp band is not visible on these gels but causes the shift in the singly-cleaved product to 645 bp. (E) Genomic sequence analysis of TALEN clones. TALEN generated insertions (lower case letters) and deletions are indicated in relation to the WT nucleotide and amino acid sequences. AseI restriction sites are boxed (yellow) and in-frame stop codons are highlighted (red) and identified with a red asterisk.DOI:http://dx.doi.org/10.7554/eLife.04137.003
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Gene editing the FCHO2 locus in HeLa cells.(A) Domain arrangement of Homo sapiens (Hs) and S. cerevisiae (Sc) muniscin family proteins. The crescent-shaped EFC domain (Extended FCH (Fer/Cip4 homology) domain) is alternatively designated the F-BAR domain due to an overall structural homology of the α-helical anti-parallel EFC dimer to the BAR (Bin1/amphiphisin/Rvs) domain family of proteins. (B) Chromosomal location and genomic organization of the FCHO2 gene with pertinent details of TALEN design. The repeat variable di-residues (RVD) selective for the different deoxyribonucleotides are color-coded (single letter amino acid notation). The endogenous AseI recognition sequence within the targeted exon is boxed (yellow). (C) Gene-specific RT-PCR analysis of various endocytic protein and control mRNA transcripts in the parental HeLa SS6 and neuroblastoma SH-SY5Y cells. HC; heavy chain. (D) AseI restriction enzyme digestion of FCHO2 gene-specific PCR amplicons from genomic DNA extracted from wild-type (WT) and TALEN-treated clones. The undigested parental (HeLa) PCR product and digested PCRs are shown. The ‘pool’ designates a PCR reaction from a genomic DNA sample of TALEN-transfetced HeLa cells prior to clone selection. The AseI nuclease generates three PCR DNA fragments; the 55-bp band is not visible on these gels but causes the shift in the singly-cleaved product to 645 bp. (E) Genomic sequence analysis of TALEN clones. TALEN generated insertions (lower case letters) and deletions are indicated in relation to the WT nucleotide and amino acid sequences. AseI restriction sites are boxed (yellow) and in-frame stop codons are highlighted (red) and identified with a red asterisk.DOI:http://dx.doi.org/10.7554/eLife.04137.003
Mentions: FCHO1 (KIAA0290; located on chromosome 19) and FCHO2 (on chromosome 5) display ∼50% overall amino acid identity and a similar domain organization (Figure 1A; Katoh, 2004). Both are EFC domain proteins (Shimada et al., 2007) characterized by a C-terminal μ-homology domain (μHD), distantly related in primary sequence but with an analogous fold to the cargo-engaging μ2 subunit of the heterotetrameric AP-2 clathrin adaptor (Reider et al., 2009). This combination of an EFC domain (alternatively designated the F-BAR domain [Frost et al., 2008; Heath and Insall, 2008]) with a μHD is unique, and these paralogous proteins have orthologues in chordates, arthropods and nematodes (Katoh, 2004; Umasankar et al., 2012; Mayers et al., 2013), as well as in unicellular eukaryotes (Boettner et al., 2009; Reider et al., 2009; Stimpson et al., 2009). In the budding yeast Saccharomyces cerevisiae, Syp1p has an analogous architecture to FCHO1/2 despite limited (<20%) overall primary sequence identity (Figure 1A). Phylogenetic dendrograms (TreeFam TF328986) (Ruan et al., 2008) show that a third chordate member, Sgip1, is more closely related to FCHO2 than to FCHO1, but this neuronal protein, while containing a modular C-terminal μHD, lacks the folded N-terminal helical EFC domain (Figure 1A; Uezu et al., 2007; Dergai et al., 2010). At steady state, endogenous FCHO2 (Henne et al., 2010; Umasankar et al., 2012) and both transfected FCHO1 (Reider et al., 2009; Taylor et al., 2011; Umasankar et al., 2012) and Sgip1 (Uezu et al., 2007; Stimpson et al., 2009) colocalize extensively with clathrin-coated structures, unlike several other EFC domain proteins (Cip4/Fbp17/Pacsin) implicated in endocytosis (Modregger et al., 2000; Kamioka et al., 2004; Taylor et al., 2011).10.7554/eLife.04137.003Figure 1.Gene editing the FCHO2 locus in HeLa 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