A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing
- University of Pittsburgh School of Medicine, United States
- Washington University School of Medicine, United States
Figures
Figure 1
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 …
Figure 2
FCHO2-nullizygous HeLa cells exhibit abnormal clathrin-coated structures at the plasma membrane.
(A) Whole cell lysates from wild-type (WT) HeLa SS6 cells and TALEN clones #6, #27, #52, #64, #68 and #71 were analyzed by SDS-PAGE. Duplicate immunoblots were probed with polyclonal antibodies …
Figure 3
Characterization of FCHO2-null 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) …
Figure 4 with 1 supplement
TALEN targeting of the FCHO1 locus in HeLa clone #64 cells.
(A) Chromosomal location and genomic organization of the FCHO1 gene with pertinent details of TALEN design. The repeat variable diresidues (RVD) selective for the different deoxynucleotides are …
Figure 4—figure supplement 1
FCHO1 transcript silencing does not alter the arrangement of surface clathrin-coated structures.
(A–C) HeLa SS6 cells were either mock transfected (A) or transfected with siRNA oligonucleotides directed against the FCHO1 (B) or FCHO2 (C) mRNA (Umasankar et al., 2012). Silenced cells were fixed …
Figure 5
HeLa clone #64/1.E cells have undetectable levels of muniscin proteins.
(A) Samples of 100 μg of GST or GST-EPS15 (595–896) prebound to glutathione-Sepharose beads were incubated with cell lysates from HeLa SS6, clone #64, clone #64/1.E and K562 cells. After washing, …
Figure 6 with 1 supplement
Ultrastructural analysis of gene-edited clone #64/1.E cell clathrin lattices.
(A–E) Selected but representative deep etch-EM replicas revealing the glass-attached ventral surface of control HeLa SS6 (A and B) or clone #64/1.E (C–E) cells. Polyhedral clathrin assemblies are …
Figure 6—figure supplement 1
Lattice assembly defects in the FCHO1/2-depleted HeLa clone #64/1.E cell line.
(A–D) Similarly sized planar clathrin lattice regions from freeze-etch replicas of HeLa SS6 control (A and B) or HeLa clone #64/1.E cells (C and D) representative of the population of …
Figure 7 with 1 supplement
Clathrin-dependent cargo internalization in HeLa clone #64/1.E cells.
(A and B) Representative confocal optical sections of HeLa SS6 (A) or clone #64/1.E (B) cells incubated with 25 μg/ml Alexa Fluor488-conjugated transferrin (green) for 2 min at 37°C before washing …
Figure 7—figure supplement 1
Reconstitution of transferrin capture within clathrin-coated structures in HeLa clone #64/1.E cells.
(A–C) HeLa clone 1.E cells transiently transfected with full-length GFP-FCHO1 (1–889) (green) were observed by total internal fluorescence microscopy after addition of 25 μg/ml Alexa Fluor568 …
Figure 8 with 2 supplements
The muniscin unstructured linker sector regulates clathrin-coated structure topology.
(A–H′) Representative single confocal optical sections of HeLa clone #64/1.E cells transiently transfected with GFP (A and A′), GFP-FCHO1 (1–889) (B and B′), GFP-FCHO1 EFC domain (1–275) (C and C′), …
Figure 8—figure supplement 1
Comparative expression of GFP-tagged FCHO1 protein fragments in HeLa cells.
Total cell lysates from HeLa SS6 cells transiently transfected with the indicated GFP-tagged FCHO1 protein fusions were resolved by SDS-PAGE and transferred in duplicate to nitrocellulose. Blots …
Figure 8—figure supplement 2
Oversized and clumped clathrin-coated structures in MCF-7 cells are normalized by ectopic FCHO1 or FCHO2 expression.
(A–D). MCF-7 cells transiently transfected with either fill-length GFP-tagged FCHO1 (residues 1–889) (A), GFP-FCHO2 (residues 1–810) (B), GFP-FCHO1 EFC + linker (residues 1–609) (C) or GFP-FCHO1 EFC …
Figure 9 with 1 supplement
Functionally significant phylogenetic conservation within the muniscin central linker domain.
(A) Muniscin–AP-2 interactions. T-Coffee (Notredame et al., 2000) generated multiple sequence alignment of the phylogenetically conserved linker region within muniscin members. Amino acid regions of …
Figure 9—figure supplement 1
Cytosolic AP-2 and the Necap 1 PHear domain (residues 1–133) bind to FCHO1 and FCHO2.
(A) Samples of ∼200 μg of GST, GST-FCHO1 (316–467), GST-FCHO2 (314–740) or GST-Sgip1 (77–214) immobilized on glutathione-Sepharose were used in pull-down assays as in with rat brain cytosol as in Fig…
Figure 10
Trafficking of exogenous Tac and Tac-FCHO1 fusion proteins in HeLa cells.
(A–A′) Maximal projection (A) and a selected medial plane (A′) of deconvolved confocal image z-stacks of HeLa SS6 cells transiently transfected with a Tac-encoding plasmid. Prior to fixation, the …
Figure 11
An artificial transmembrane FCHO1 linker protein misrecruits AP-2 onto internal membrane structures.
(A–A″) Selected medial (A and A′) or basal (A″) optical sections of deconvolved confocal z-stacks collected from HeLa SS6 cells transiently transfected with Tac-FCHO1 linker (residues 265–609). …
Figure 12
PtdIns(4,5)P2 is not enriched at sites of intracellular AP-2 accumulation.
(A–F) HeLa SS6 cells cotransfected with a mixture of either Tac-FCHO1 linker (residues 265–609) (A–C) or Tac-FCHO1 linker + μHD (residues 265–889) (D–F) and GFP-PLCδ1 PH domain encoding plasmids …
Figure 13 with 1 supplement
Forced expression of the Tac-FCHO1 linker fusion restores clathrin coat distribution in gene-edited HeLa cells.
(A–P) Basal (A, E, I, M) and medial (B, F, J, N) confocal sections from deconvolved z-image stacks from HeLa clone #64/1.E cells immunolabeled with antibodies against AP-2 (mAb AP.6; green) and …
Figure 13—figure supplement 1
Mislocalized EPS15 in Tac-linker expressing HeLa clone #64/1.E cells overlaps with intracellular Tac.
(A–D) HeLa clone #64/1.E cells were short-term transfected with Tac (A), Tac-FCHO1 linker (residues 265–609) (B), Tac-FCHO1 μHD (residues 609–889) (C) or Tac-FCHO1 linker + μHD (residues 265–889) (D)…
Figure 14
Direct regulation of coat morphology and cargo packaging by the FCHO1 linker domain.
(A and B) Color channel separated total internal reflection images of HeLa SS6 cells stably expressing β2-YFP (A) and pulsed for 2 min with 25 μg/ml Alexa Fluor647 transferrin (B). The cells were …
Author response image 1
Additional files
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Supplementary file 1
List of various constructs used in this study and the sets of specific primers, restriction sites, plasmids and the methods of cloning used to design these constructs.
- https://doi.org/10.7554/eLife.04137.024
