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Developmental and morphological regulation of clathrin-mediated endocytosis in Trypanosoma brucei

Gareth W. Morgan1, Clare L. Allen1, Tim R. Jeffries1, Michael Hollinshead2 and Mark C. Field1,*

1 Wellcome Trust Laboratories for Molecular Parasitology, Imperial College of Science Technology and Medicine, Department of Biochemistry, Exhibition Road, London, SW7 2AY, UK
2 Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
* Author for correspondence (e-mail: m.field{at}ic.ac.uk )



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  		Fig. 1. Sequence features of trypanosome clathrin and adaptin. (A) The clathrin heavy chain. Structural domains of T. brucei (TbCLH) are depicted as determined on the basis of similarity to human CLH1 (accession no. Q00610) and yeast CLH (accession no. P22137). GH, globular head; L, flexible linker; DA, distal arm; PA, proximal arm; LC, light chain binding region; TR, trimerisation region. Percentage identity/similarity to yeast CLH (Sc) and human CLH1 (Hs) are given below each domain. The relative locations of each sheared T. brucei genomic DNA sequence (obtained from the TIGR T. brucei genome sequencing project website: www.tigr.org/tdb/mdb/tbdb) and PCR fragments used to compile the complete TbCLH open reading frame are depicted below. Thin lines represent the locations of sheared genomic DNA end sequences and the broken line represents the end sequence of a BAC genomic DNA clone. Accession numbers are shown alongside each sequence. Thick lines represent the locations of the PCR fragments. (B) The ß-adaptin/ß-arrestin and light chain binding sites of TbCLH are conserved. The upper alignment of human, yeast and trypanosomal clathrin heavy chain shows the high degree of conservation within the ß-adaptin and ß-arrestin binding site. The residues Q89, F91, K96 and K98 critical for arrestin binding by human clathrin heavy chain are conserved. The lower alignment shows the conservation of residues involved in light chain binding. Large arrows indicate residues involved in trimerisation and light chain binding; small arrows indicate residues preferentially involved in light chain binding. Identical residues are boxed, conservative mutations are shaded. (C) Schematic representation of the domain structure of human ß1-adaptin and TbAPß1. Higher eukaryotic ß-adaptins are comprised of a 60-70 kDa trunk domain separated from a 25-30 kDa appendage domain by a ~100 residue linker. The appendage domain is absent in the TbAPß1 sequence. The binding sites for arrestins, clathrin heavy chain (CHC), AP180, epsin and eps15 in higher eukaryotic ß-adaptin are illustrated. Also shown is a putative clathrin binding box sequence in TbAPß1. Trypanosomal and yeast ß1-adaptins terminate with a sequence similar to clathrin binding sequences located in the hinge region of human ß-adaptins (HuAPß1,2,3) and the C-termini of a yeast epsin homologue (ScEnt) and yeast AP180 (ScAP180). The highly conserved leucine and aspartate residues are boxed.

 


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  		Fig. 2. Conservation of the clathrin and adaptin from T. brucei. The evolutionary relationship of TbCLH and TbAPß1 was analysed using PAUP. TbCLH is demonstrated to be most closely related to the heavy chain of S. cerevisiae (A) and TbAPß1 is most homologous to S. cerevisiae ß1-adaptin (B). Numbers represent the bootstrap percent confidence for various branch points and horizontal distances represent relative genetic distance. Vertical distances are for clarity only. The ß-adaptin sequences used are: Human (Hs) APß1, ß2, ß3, ß4; Yeast (Sc) APß1 (apl2p), ß2 (apl1p), ß3 (apl6p); A. thaliana (At APß); and T. brucei ß1 (Tb APß1).

 


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  		Fig. 3. Trypanosome clathrin and adaptin mRNAs developmentally regulated. Northern blot analysis of TbCLH and TbAPß1 mRNAs. Total RNA from BSF and PCF cells was hybridised at high stringency with a trypanosomal clathrin heavy chain or ß1-adaptin specific probe. Scale at the left represents relative size in kb; ethidium bromide stain of the ribosomal RNA region at the bottom indicates equivalence of loading.

 


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  		Fig. 4. Developmental regulation of trypanosomal clathrin and adaptin expression at the protein level. Western blot analysis demonstrates developmental regulation of TbCLH protein. (A) Antibodies generated against a fragment of TbCLH (residues 1268-1465) exhibit specific reactivity against the protein, as demonstrated by immunoprobing E. coli lysate expressing (+) or not expressing (-) a fragment of TbCLH (left). Immunoprobing of 1x107 parasites with anti-TbCLH antibodies demonstrates that the heavy chain is expressed at much higher levels in BSF than PCF cells (right). Trypanosomal binding protein (BiP) is used as a loading control. (B) Reactivity of the anti-peptide antibodies to TbAPß1 was confirmed by immunoprobing yeast expressing TbAPß1. Equal lysates (~2x107 cell equivalents) from untransformed or transformed S. cerevisiae strain GPY418 with pGADT7ßAd were loaded per lane. The left and middle panels show yeast expressing (+) or not expressing (-) HA epitope-tagged TbAPß1 probed with anti-TbAPß1 antibody (left) or anti-HA antibody (middle). Immunoprobing trypanosomal BSF or PCF lysate with anti-TbAPß1 antibodies demonstrate approximate equivalence in protein levels (right). Scale at the left represents molecular mass in kDa (A,B).

 


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  		Fig. 5. Localisation of TbCLH and TbAPß1 in different life stages. The far-left panels show PCF cells (A,C) or BSF cells (B,D) probed with anti-TbCLH (A,B) or anti-TbAPß1 antibodies (C,D). The centre-left panels show DAPI staining, and the centre-right panels show phase-contrast of the cells. The position of the antibody labelling relative to the nucleus and the kinetoplast is shown in the merged panels (far-right). In PCF, TbCLH is localised to discrete punctata between the nucleus and the kinetoplast but is widely distributed on large membraneous structures throughout the posterior end of the BSF. In the PCF, TbAPß1 is present on several small peri-nuclear vesicles and reticular elements (C) but, by contrast, is concentrated in two large perinuclear structures in the BSF (D).

 


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  		Fig. 6. TbCLH and TbAPß1 closely associate with the Golgi complex, but are on distinct membranes. (A,B) BSF cells were stained with TbCLH (A, left) or TbAPß1 (B, left), BODIPY-ceramide (middle), and DAPI (in all panels); the merged image is shown on the right. TbCLH is observed in structures juxtaposed to the Golgi complex, whereas the TbAPß1 membranous structure nearest to the nucleus partially co-localises to the Golgi complex as seen in yellow in the merged image. (C,D) PCF (C) and BSF (D) cells labelled with rabbit anti-TbCLH (red) and mouse anti-TbAPß1 (green) and examined by confocal microscopy. The merged image is shown on the right; the asterix indicates the position of the nucleus. In neither of these life stages is there significant coincidence.

 


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  		Fig. 7. Differential localisation of TbCLH during the cell cycle. TbCLH is closely associated with the kinetoplast in the PCF and as the kinetoplast divides during mitosis, the pool of TbCLH is also observed to divide and separate with this structure. This behaviour is consistent with the vast majority of TbCLH being associated with membranes subtending the flagellar pocket. The distribution of TbCLH during the cell cycle in BSF is also shown; again TbCLH infills following kinetoplast division, but is less clear cut due to the extensive clathrin network in this life stage.

 


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  		Fig. 8. ConA and transferrin uptake demonstrate TbCLH is involved in endocytosis. BSF cells were pulsed with fluorescein-ConA for 30 seconds (A) or 1 minute (B), or with Texas Red-transferrin (C). The localisation of TbCLH is shown in the left panel, ConA or transferrin in the middle panel, and the merged image in the right panel. The position of the nucleus is shown by DAPI staining (blue) or marked by an asterix in C. After 1 minute, there is strong co-localisation between the lectin and TbCLH. There is a high degree of transferrin/TbCLH co-localisation, indicating that clathrin is present on structures receiving cargo targeted to endosomes by receptor-mediated endocytosis.

 


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  		Fig. 9. Ultra thin cryosections showing subcellular localisation of TbCLH. (A) Conventional Epon section showing clathrin-like coats on a vesicle (arrowheads) and a tubular profile (arrows). (B-D) Cryosections labelled with TbCLH followed by 6 nm protein A gold show clathrin in association with a tubular profile (B); on membranes and vesicles in a region between the Golgi and the flagellar pocket (C); and also TbCLH labelling of membranes and vesicles just below the plasma membrane (D). In C, the 6 nm gold particles originally used for increased resolution have been digitally enhanced using Adobe Illustrator by placing black circles corresponding to ~ 15nm diameter over the Gold particles. Bars, 100 nm, the position of the Golgi complex, endoplasmic reticulum (ER) and plasma membrane (PM) are indicated.

 

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© The Company of Biologists Ltd 2001