spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 28 February 2006
doi: 10.1242/jcs.02809

Journal of Cell Science 119, 1105-1117 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Helms, M. J.
Right arrow Articles by Mottram, J. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Helms, M. J.
Right arrow Articles by Mottram, J. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Bloodstream form Trypanosoma brucei depend upon multiple metacaspases associated with RAB11-positive endosomes

Matthew J. Helms1,*, Audrey Ambit1,*, Paul Appleton2, Laurence Tetley2, Graham H. Coombs2 and Jeremy C. Mottram1,{ddagger}

1 Wellcome Centre for Molecular Parasitology, The Anderson College, University of Glasgow, Glasgow G11 6NU, UK
2 Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK


Figure 1
View larger version (16K):

[in a new window]
  		Fig. 1. Expression of metacaspases. (A,B) Total cell lysates were prepared from T. brucei PCF (lane 1) and BSF (lane 2). 5x106 cell equivalents were then subjected to SDS-PAGE and transferred to PVDF membrane prior to immunoblotting with immunopurified rabbit anti-MCA2-MCA3 antibodies (A) or with sheep anti-MCA5 antiserum (B). MCA2, MCA3 and MCA5 are indicated. A crossreacting 48 kDa protein (A, see *) and an antibody against EF-1{alpha} demonstrates equal loading. (C) Western blots prepared with BSF wild-type (lane 1) and transgenic parasites expressing an ectopic copy of either MCA3HA (lane 2) or MCA2HA (lane 3) were probed with a monoclonal anti-HA antibody. MCA2HA and MCA3HA are indicated at their predicted sizes of 40 and 41 kDa, respectively.

 

Figure 2
View larger version (42K):

[in a new window]
  		Fig. 2. RNAi of metacaspases in BSF T. brucei. (A). Western blot of cell extracts from an MCA2-MCA3 RNAi clone (left panel) or an MCA5 RNAi clone (right panel), incubated with (+) or without (-) tetracycline (tet) and probed with anti-MCA2-MCA3 or anti-MCA5 antibodies. Asterisk indicates crossreacting 48 kDa protein. (B) Upper panel, growth curve of an MCA2-MCA3 and MCA5 triple RNAi clone with or without induction with tetracycline. Lower panels, western blot of cell extracts from an MCA2-MCA3 and MCA5 triple RNAi clone, with or without induction with tetracycline, probed with anti-MCA2-MCA3 (left) or anti-MCA5 antibodies (right). (C) Upper panel, normal cell-cycle progression in wild-type BSF T. brucei. The kinetoplast division occurs before nuclear division, ultimately followed by cytokinesis. Images illustrate various cell stages with DAPI-stained nuclei (N) and kinetoplasts (K) (white); insets show kinetoplast at higher magnification (blue). Lower panels show nucleus and kinetoplast configurations with or without triple RNAi induction with tetracyline. Others, cells with an abnormal configuration but excluding 2N1K cells. For each time point, 200 cells were analysed. (D) Upper panel, analysis of kinetoplast configuration in 1N1K cells after induction with tetracycline. Bottom panels, aberrant cell-cycle progression in MCA2-MCA3-MCA5 RNAi-induced BSF T. brucei. Images of representative triple RNAi-induced BSF T. brucei. Inset shows kinetoplast at higher magnification. Bars, 10 µm.

 

Figure 3
View larger version (25K):

[in a new window]
  		Fig. 3. Generation of metacaspase null mutants. (A) Schematic representation of the MCA2-MCA3 and MCA5 loci together with constructs for gene knockout. Open reading frames (ORFs) are shown as arrows, flanking DNA sequences for targeting are shown as boxes. The predicted fragment sizes of KpnI-digested DNA for both native and modified MCA2-MCA3 locus is shown. BSD, blasticidin-resistance gene; PAC, puromycin-resistance gene; HYG, hygromycin-resistance gene; NEO, neomycin-resistance gene. (B) Southern blot analysis of {Delta}mca2/3. Genomic DNA was digested with KpnI, separated on a 1% agarose gel, blotted onto Hybond-P membrane and hybridised with a 32P-labelled DNA probe comprising the 5' flanking region used in the knockout strategy. Lane 1, wild type; lane 2, PAC heterozygote; lane 3, BSD heterozygote; lane 4, {Delta}mca2/3 clone 1; lane 5, {Delta}mca2/3 clone 2. (C) Total cell lysates were prepared from BSF parasites and 5x106 cell equivalents subjected to SDS-PAGE and transferred to PVDF membrane prior to immunoblotting with rabbit anti-MCA2-MCA3 antibodies. Lane 1, wild type; lane 2, {Delta}mca2/3; lane 3, {Delta}mca2/3{Delta}mca5; lane 4, {Delta}mca2/3{Delta}mca5:MCA3; lane 5, {Delta}mca2/3{Delta}mca5:MCA2. MCA2 and MCA3 are indicated. Asterisk indicates crossreacting 48 kDa protein. (D) Total cell lysates were prepared from BSF parasites and 5x106 cell equivalents subjected to SDS-PAGE and transferred to PVDF membrane prior to immunoblotting with sheep anti-MCA5 antiserum. Lane 1, wild type; lane 2, {Delta}mca5; lane 3, {Delta}mca2/3{Delta}mca5; lane 4, {Delta}mca2/3{Delta}mca5:MCA5. MCA5 is indicated.

 

Figure 4
View larger version (22K):

[in a new window]
  		Fig. 4. Location of metacaspases. (A) BSF T. brucei parasites expressing either MCA2HA or MCA3HA proteins were fixed and co-stained with anti-HA monoclonal antibody (red) and sheep anti-MCA5 antiserum (green). (B) BSF parasites were fixed and stained with rabbit anti-RAB11 (red) and sheep anti-MCA5 antiserum (green). Single-slice images were analysed with volume deconvolution by using nearest-neighbour algorithm. (C) BSFs were incubated with rabbit anti-VSG221 antiserum at 4°C for 30 minutes and then returned to 37°C for 30 minutes. Fixed and permeabilised cells were stained with anti-MCA5 antiserum (green) and anti-rabbit IgG (red). For all images DNA was visualised with DAPI (blue). DIC images of the parasite are shown. Colocalisation is shown in yellow. Bars, 10 µm.

 

Figure 5
View larger version (213K):

[in a new window]
  		Fig. 5. Immunogold transmission-electron microscopy for BSF T. brucei. (A) Two vesicles labelled, one distinct MCA5 only (10 nm gold rabbit anti-sheep antibody) and one with RAB11 (6 nm gold goat anti-rabbit antibody) and MCA5. The particle groupings indicate an underlying bounding structure in the cytoplasm. (B) Presence of 10-nm and 6-nm gold particles at the periphery of a compartment. White arrowheads indicate bounding membrane. (C) Further example of RAB11 staining close to the flagellar pocket (D) Localisation of MCA5 only (white arrowhead), RAB11 with MCA5 (white arrow) and RAB11 only (black arrowhead) in T. brucei. Black arrow indicates surface coat; fp, flagellar pocket. Bars, 50 nm.

 

Figure 6
View larger version (64K):

[in a new window]
  		Fig. 6. Recycling of VSG. Wild-type, {Delta}mca2/3{Delta}mca5 and triple RNAi lines, with or without induction with tetracycline for 8 hours. Parasites were biotinylated at 4°C prior to incubation at 37°C for 5 minutes in HMI-9 medium. Remaining surface label was then cleaved by incubation with 50 mM glutathione at 4°C. Cells were prepared for fluorescence microscopy both immediately after the glutathione incubation and following a 5- or 10-minute chase period in HMI-9 medium at 37°C. Parasites were stained with Texas-Red-conjugated streptavidin (red). DNA was visualised with DAPI (blue). Insets, DIC images of the parasite. Bars, 10 µm.

 

Figure 7
View larger version (43K):

[in a new window]
  		Fig. 7. Degradation of anti-VSG IgG. BSF wild-type, {Delta}mca2/3{Delta}mca5 and triple RNAi lines, with or without induction with tetracycline for 8 hours. Parasites were labelled with anti-VSG221 IgG at 4°C in HMI-9 prior to incubation at 37°C for 5 and 10 minutes in HMI-9. (A) Parasites were fixed and stained with anti-rabbit-FITC conjugate (green) and DAPI (blue). Insets, DIC images of the parasite. Bars, 10 µm. (B) Whole-cell lysates were prepared after the 30-minute incubation and 5x106 cell equivalents subjected to SDS-PAGE and transferred to PVDF membrane prior to immunoblotting with an HRP-conjugated anti-rabbit IgG. Lane 1, wild-type BSF; lane 2, {Delta}mca2/3{Delta}mca5. EF-1{alpha} served as a loading control. (C) TCA precipitations of the culture medium were resuspended, subjected to SDS-PAGE and transferred to PVDF membrane prior to immunoblotting with an HRP-conjugated anti-rabbit IgG. Lane 1, without anti-VSG221 IgG; lane 2, with anti-VSG221 IgG in the absence of parasites; lane 3, wild-type BSF; lane 4, {Delta}mca2/3{Delta}mca5 BSF. Anti-VSG221 IgGs are indicated together with degradation products.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006