Developmental and morphological regulation of clathrin-mediated endocytosis inTrypanosoma brucei

2001 ◽  
Vol 114 (14) ◽  
pp. 2605-2615 ◽  
Author(s):  
Gareth W. Morgan ◽  
Clare L. Allen ◽  
Tim R. Jeffries ◽  
Michael Hollinshead ◽  
Mark C. Field
Keyword(s):  
Heavy Chain ◽  
Developmental Regulation ◽  
Bloodstream Form ◽  
Host Immune System ◽  
Coat Proteins ◽  
Flagellar Pocket ◽  
Coated Pits ◽  
Ultrastructural Studies ◽  
Clathrin Heavy Chain ◽  
Collecting Tubules

Essentially all macromolecular communication between Trypanosoma brucei and its host is confined to vesicular trafficking events occurring at or around the flagellar pocket. The vertebrate stage bloodstream form trypomastigote exhibits an extremely high rate of endocytosis required for nutrient uptake and probably also evasion of the host immune system. However, the rate of endocytosis is very low in the procyclic vector parasite, indicating that endocytosis is subject to a marked level of developmental regulation. Previous ultrastructural studies and crude biochemical fractionations have indicated the presence of coated pits and vesicles that are analogous to clathrin coats in the bloodstream form, but not in the procyclic. However, a definitive description of the components of this coat and its molecular function in T. brucei has remained elusive. We describe the molecular cloning and initial characterisation of components of the T. brucei endocytic coats: clathrin heavy chain (TbCLH) and a β-adaptin (TbAPβ1). TbCLH is markedly upregulated in the bloodstream form compared with the procyclic, whereas TbAPβ1 is subject to more limited developmental regulation. We generated antisera against both proteins and show that the clathrin coat is tightly associated with the flagellar pocket in both major life stages. However, in bloodstream parasites TbCLH is also extensively distributed throughout the posterior end of the cell on numerous large vesicular and tubular structures. By cryoimmuno EM, clathrin is localised to collecting tubules at the flagellar pocket and is also associated with the trans-Golgi network. These EM data confirm that the electron dense coats reported on trypanosome vesicles and tubules contain clathrin. The TbAPβ1 exhibits an atypical distribution relative to previously characterised adaptins, associating not only with the trans-Golgi but also with other tubular-vesicular elements. Localisation of TbAPβ1 is also subject to developmental regulation. These data describe major endocytic coat proteins in T. brucei for the first time, and indicate stage-specific expression of the clathrin heavy chain. Modulation of clathrin expression is likely to be an important factor in the developmental regulation of endocytosis and recycling in the African trypanosome.

Structure of clathrin cages and coats in ice

1986 ◽  
Vol 44 ◽  
pp. 94-97
Author(s):  
G.P.A. Vigers ◽  
R.A. Crowther ◽  
B.M.F. Pearse
Keyword(s):  
Electron Microscopy ◽  
Heavy Chain ◽  
Outer Part ◽  
Coated Vesicles ◽  
Eukaryotic Cells ◽  
Coat Proteins ◽  
Terminal Domain ◽  
Clathrin Heavy Chain ◽  
Membrane Components

Clathrin forms the polyhedral cage of coated vesicles, which mediate the transfer of selected membrane components within eukaryotic cells. Clathrin cages and coated vesicles have been extensively studied by electron microscopy of negatively stained preparations and shadowed specimens. From these studies the gross morphology of the outer part of the polyhedral coat has been established and some features of the packing of clathrin trimers into the coat have also been described. However these previous studies have not revealed any internal details about the position of the terminal domain of the clathrin heavy chain, the location of the 100kd-50kd accessory coat proteins or the interactions of the coat with the enclosed membrane.

scholarly journals Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor μ2 kinase

2003 ◽  
Vol 163 (2) ◽  
pp. 231-236 ◽  
Author(s):  
Antony P. Jackson ◽  
Alexander Flett ◽  
Carl Smythe ◽  
Lindsay Hufton ◽  
Frank R. Wettey ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Heavy Chain ◽  
Transferrin Receptor ◽  
Coated Pits ◽  
Permeabilized Cells ◽  
Clathrin Heavy Chain

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.

Internalization of cholera toxin by different endocytic mechanisms

2001 ◽  
Vol 114 (20) ◽  
pp. 3737-3747 ◽  
Author(s):  
Maria L. Torgersen ◽  
Grethe Skretting ◽  
Bo van Deurs ◽  
Kirsten Sandvig
Keyword(s):  
Cholera Toxin ◽  
Heavy Chain ◽  
Large Fraction ◽  
Cell Types ◽  
Coated Pits ◽  
Bhk Cells ◽  
Clathrin Heavy Chain ◽  
Antisense Mrna ◽  
Inducible Synthesis ◽  
Different Cell Types

The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cells in which antisense mRNA to clathrin heavy chain can be induced. Here we show that endocytosis and the ability of CT to increase the level of cAMP were unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner. Treatment of Caco-2 cells with filipin reduced CT-uptake by less than 20%, suggesting that caveolae do not play a major role in the uptake. Extraction of cholesterol by methyl-β-cyclodextrin, which removes caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% reduction of CT-endocytosis. Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis. These cells contain few caveolae, and nystatin and filipin had no effect on CT-uptake, indicating major involvement of clathrin-coated pits in CT-internalization. Similarly, in BHK cells, where clathrin-dependent endocytosis is blocked by induction of antisense clathrin heavy chain, the CT-uptake was reduced by 50% in induced cells. In conclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different cell types.

A developmentally regulated Rab11 homologue in Trypanosoma brucei is involved in recycling processes

2001 ◽  
Vol 114 (14) ◽  
pp. 2617-2626 ◽  
Author(s):  
Tim R. Jeffries ◽  
Gareth W. Morgan ◽  
Mark C. Field
Keyword(s):  
Trypanosoma Brucei ◽  
Basal Body ◽  
Transferrin Receptor ◽  
Developmental Regulation ◽  
Early Endosome ◽  
Surface Glycoprotein ◽  
Flagellar Pocket ◽  
Developmentally Regulated ◽  
Regulated Expression ◽  
Collecting Tubules

Endocytosis in the parasitic protozoan Trypanosoma brucei, a deeply divergent eukaryote, is implicated as important in both general cellular function and virulence, and is strongly developmentally regulated. We report the characterisation of a previously undefined endosomal compartment in T. brucei based on identification of a new trypanosome gene (TbRAB11) homologous to Rab11/Ypt31. Northern and western analyses indicated that TbRAB11 expression was significantly upregulated in the bloodstream stage of the parasite, the first trypanosome Rab to be identified with a developmentally regulated expression profile. In procyclic form parasites TbRAB11 localised to a compartment positioned close to the basal body, similar to mammalian Rab11. By contrast, in bloodstream form parasites, TbRAB11-containing structures were more extensive and the TbRAB11 compartment extended towards the posterior face of the nucleus, was more elaborate and was not always adjacent to the basal body. Colocalisation studies by light and confocal microscopy demonstrated that TbRAB11 was located on a compartment that did not correspond to other established trypanosomal organelles or markers. Using concanavalin A internalisation and temperature block procedures, TbRAB11 was observed on endomembranes anterior to the flagellar pocket that are juxtaposed to the collecting tubules. TbRAB11 colocalised with the trypanosomal transferrin receptor and internalised antivariant surface glycoprotein. Further, we show that the collecting tubules contain TbRAB5A, suggesting that they are the trypanosomatid early endosome. Hence, TbRAB11 is present on endosomal structures that contain recycling cargo molecules and is under developmental regulation, suggesting a role in stage-dependent endocytic processes.

scholarly journals SCD5, a suppressor of clathrin deficiency, encodes a novel protein with a late secretory function in yeast.

1996 ◽  
Vol 7 (2) ◽  
pp. 245-260 ◽  
Author(s):  
K K Nelson ◽  
M Holmer ◽  
S K Lemmon
Keyword(s):  
Heavy Chain ◽  
Secretory Pathway ◽  
Vesicular Transport ◽  
Carboxypeptidase Y ◽  
Chain Gene ◽  
Temperature Sensitive ◽  
Coat Proteins ◽  
Clathrin Heavy Chain ◽  
Biochemical Analyses ◽  
Novel Protein

Clathrin and its associated proteins constitute a major class of coat proteins involved in vesicle budding during membrane transport. An interesting characteristic of the yeast clathrin heavy chain gene (CHC1) is that in some strains a CHC1 deletion is lethal, while in others it is not. Recently, our laboratory developed a screen that identified five multicopy suppressors that can rescue lethal strains of clathrin heavy chain-deficient yeast (Chc - scd1-i) to viability. One of these suppressors, SCD5, encodes a novel protein of 872 amino acids containing two regions of repeated motifs of unknown function. Deletion of SCD5 has shown that it is essential for cell growth at 30 degrees C. scd5-delta strains carrying low copy plasmids encoding C-terminal truncations of Scd5p are temperature sensitive for growth at 37 degrees C. At the nonpermissive temperature, cells expressing a 338-amino acid deletion (Scd5P-delta 338) accumulate an internal pool of fully glycosylated invertase and mature alpha-factor, while processing and sorting of the vacuolar hydrolase carboxypeptidase Y is normal. The truncation mutant also accumulates 80- to 100-nm vesicles similar to many late sec mutants. Moreover, at 34 degrees C, overexpression of Scd5p suppresses the temperature sensitivity of a sec2 mutant, which is blocked at a post-Golgi step of the secretory pathway. Biochemical analyses indicate that approximately 50% of Scd5p sediments with a 100,000 x g membrane fraction and is associated as a peripheral membrane protein. Overall, these results indicate that Scd5p is involved in vesicular transport at a late stage of the secretory pathway. Furthermore, this suggests that the lethality of clathrin-deficient yeast can be rescued by modulation of vesicular transport at this late secretory step.

scholarly journals Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium.

1992 ◽  
Vol 118 (6) ◽  
pp. 1371-1377 ◽  
Author(s):  
T J O'Halloran ◽  
R G Anderson
Keyword(s):  
Heavy Chain ◽  
Antisense Rna ◽  
Fluid Phase ◽  
Solid Particles ◽  
Endocytic Pathway ◽  
Coated Pits ◽  
Clathrin Heavy Chain ◽  
Development Cycle ◽  
Mutant Cells

To investigate the intracellular role of the clathrin heavy chain in living cells, we have used "antisense" RNA to engineer mutant Dictyostelium discoideum cells that are severely deficient in clathrin heavy chain expression. Immunoblots stained with an anti-clathrin heavy chain antiserum revealed that mutant cells contained undetectable amounts of clathrin heavy chain protein. Similarly, Northern blots showed an absence of clathrin heavy chain mRNA. Clathrin heavy chain-deficient Dictyostelium cells were viable, but exhibited growth rates twofold slower than parental cells. Whereas many morphological features of the mutant cells were normal, mutant cells lacked coated pits and coated vesicles. Clathrin-deficient cells were also missing large translucent vacuoles that serve as endosomes and contractile vacuoles. In the absence of clathrin heavy chain, mutant cells displayed three distinct functional defects: (a) impairment in endocytosis of fluid phase markers, but competence in another endocytic pathway, the phagocytosis of solid particles; (b) defects in osmoregulation; and (c) inability to complete the starvation-induced development cycle.

scholarly journals Clathrin Heavy Chain 1 Plays Essential Roles During Oocyte Meiotic Spindle Formation and Early Embryonic Development in Sheep

2021 ◽  
Vol 9 ◽  
Author(s):  
Zhe Han ◽  
Xin Hao ◽  
Cheng-Jie Zhou ◽  
Jun Wang ◽  
Xin Wen ◽  
...  
Keyword(s):  
Embryo Development ◽  
Heavy Chain ◽  
Spindle Assembly ◽  
Early Embryo ◽  
Meiotic Spindle ◽  
Coated Pits ◽  
Spindle Formation ◽  
Early Embryo Development ◽  
Clathrin Heavy Chain ◽  
First Polar Body

As a major protein of the polyhedral coat of coated pits and vesicles, clathrin molecules have been shown to play a stabilization role for kinetochore fibers of the mitotic spindle by acting as inter-microtubule bridges. Clathrin heavy chain 1 (CLTC), the basic subunit of the clathrin coat, plays vital roles in both spindle assembly and chromosome congression during somatic-cell mitosis. However, its function in oocyte meiotic maturation and early embryo development in mammals, especially in domesticated animals, has not been fully investigated. In this study, the expression profiles and functional roles of CLTC in sheep oocytes were investigated. Our results showed that the expression of CLTC was maintained at a high level from the germinal vesicle (GV) stage to metaphase II stage and that CLTC was distributed diffusely in the cytoplasm of cells at interphase, from the GV stage to the blastocyst stage. After GV breakdown (GVBD), CLTC co-localized with beta-tubulin during metaphase. Oocyte treatments with taxol, nocodazole, or cold did not affect CLTC expression levels but led to disorders of its distribution. Functional impairment of CLTC by specific morpholino injections in GV-stage oocytes led to disruptions in spindle assembly and chromosomal alignment, accompanied by impaired first polar body (PB1) emissions. In addition, knockdown of CLTC before parthenogenetic activation disrupted spindle formation and impaired early embryo development. Taken together, the results demonstrate that CLTC plays a vital role in sheep oocyte maturation via the regulation of spindle dynamics and an essential role during early embryo development.

scholarly journals The Drosophila clathrin heavy chain gene: clathrin function is essential in a multicellular organism.

Genetics ◽  
1993 ◽  
Vol 134 (4) ◽  
pp. 1119-1134 ◽  
Author(s):  
C Bazinet ◽  
A L Katzen ◽  
M Morgan ◽  
A P Mahowald ◽  
S K Lemmon
Keyword(s):  
X Chromosome ◽  
Heavy Chain ◽  
P Element ◽  
Cdna Clones ◽  
Drosophila Genome ◽  
Wild Type ◽  
Degenerate Primers ◽  
Coated Pits ◽  
Lethal Mutations ◽  
Clathrin Heavy Chain

Abstract The clathrin heavy chain (HC) is the major structural polypeptide of the cytoplasmic surface lattice of clathrin-coated pits and vesicles. As a genetic approach to understanding the role of clathrin in cellular morphogenesis and developmental signal transduction, a clathrin heavy chain (Chc) gene of Drosophila melanogaster has been identified by a combination of molecular and classical genetic approaches. Using degenerate primers based on mammalian and yeast clathrin HC sequences, a small fragment of the HC gene was amplified from genomic Drosophila DNA by the polymerase chain reaction. Genomic and cDNA clones from phage libraries were isolated and analyzed using this fragment as a probe. The amino acid sequence of the Drosophila clathrin HC deduced from cDNA sequences is 80%, 57% and 49% identical, respectively, with the mammalian, Dictyostelium and yeast HCs. Hybridization in situ to larval polytene chromosomes revealed a single Chc locus at position 13F2 on the X chromosome. A 13-kb genomic Drosophila fragment including the Chc transcription unit was reintroduced into the Drosophila genome via P element-mediated germline transformation. This DNA complemented a group of EMS-induced lethal mutations mapping to the same region of the X chromosome, thus identifying the Chc complementation group. Mutant individuals homozygous or hemizygous for the Chc1, Chc2 or Chc3 alleles developed to a late stage of embryogenesis, but failed to hatch to the first larval stage. A fourth allele, Chc4, exhibited polyphasic lethality, with a significant number of homozygous and hemizygous offspring surviving to adulthood. Germline clonal analysis of Chc mutant alleles indicated that the three tight lethal alleles were autonomous cell-lethal mutations in the female germline. In contrast, Chc4 germline clones were viable at a rate comparable to wild type, giving rise to viable adult progeny. However, hemizygous Chc4 males were invariably sterile. The sterility was efficiently rescued by an autosomal copy of the wild-type Chc gene reintroduced on a P element. These findings suggest a specialized role for clathrin in spermatogenesis.

scholarly journals An arf1Δ Synthetic Lethal Screen Identifies a New Clathrin Heavy Chain Conditional Allele That Perturbs Vacuolar Protein Transport in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (2) ◽  
pp. 577-589 ◽  
Author(s):  
Chih-Ying Chen ◽  
Todd R Graham
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heavy Chain ◽  
Protein Transport ◽  
Genetic Interaction ◽  
Synthetic Lethality ◽  
Carboxypeptidase Y ◽  
Coat Proteins ◽  
Transport Pathway ◽  
Clathrin Heavy Chain ◽  
Vacuolar Protein

Abstract ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1Δ allele and defined seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1Δ). Most of the swa mutants exhibit phenotypes comparable to arf1Δ mutants such as temperature-conditional growth, hypersensitivity to fluoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 3′ end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a significant defect in transport of carboxypeptidase Y or carboxypeptidase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not significantly affected in the chc1-5 mutant, further implicating clathrin specifically in the Golgi to vacuole transport pathway for carboxypeptidase Y.

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