QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 29 April 2008
doi: 10.1242/jcs.026344

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Motley, A. M. Articles by Hettema, E. H. Search for Related Content PubMed PubMed Citation Articles by Motley, A. M. Articles by Hettema, E. H.

Dnm1p-dependent peroxisome fission requires Caf4p, Mdv1p and Fis1p

Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK

View larger version (61K): [in this window] [in a new window]   Fig. 1. Peroxisome abundance is affected in cells lacking components of the mitochondrial fission machinery. (A) Cells expressing GFP-PTS1 fluorescence (top rows) or mito-GFP (bottom rows) were grown to log phase on 2% glucose-containing medium and representative images were captured. Images are flattened z-stacks. Scale bar: 5 µm. (B) Peroxisome numbers were counted from images of >100 budding cells for each strain.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Overexpression of Dnm1p restores peroxisome abundance in vps1/dnm1 cells, but not in vps1/fis1 or vps1/caf4/mdv1 cells. Peroxisomes were visualised using GFP-PTS1. Images are flattened z-stacks. Scale bar: 5 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Mating experiment showing that Dnm1p-mediated peroxisome fission requires the presence of Fis1p, Mdv1p and Caf4p. Fission of the pre-labelled peroxisomal structure occurs when Dnm1p is supplied by cytoplasmic mixing after mating (AxB). Fission occurs soon after cytoplasmic mixing, before zygote formation. When both mating partners are deficient for Vps1p and Dnm1p, the pre-labelled tubulated peroxisomes coexist together in the mating cells (BxC). When one of the mating partners is deficient in Fis1p or Mdv1p and Caf4p, the pre-labelled tubulated peroxisome (which is lacking Fis1p or Mdv1p/Caf4p) initially fails to divide in spite of Dnm1p being supplied in the cytoplasm of the mating partner (AxD, AxE). Fission starts to occur only when the zygote is being formed. Images are flattened z-stacks. Scale bar: 5 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Redirecting Fis1p to peroxisomes by expression of a Fis1p-Pex15p fusion protein rescues the peroxisomal fission defect in vps1/fis1 cells. Expression of Fis1p rescues the mitochondrial phenotype to that of wild-type cells, but restores the peroxisome phenotype to that of vps1 cells. Expression of Fis1p-Pex15p does not rescue the mitochondrial phenotype, but restores the peroxisome phenotype to that of wild-type cells. Images are flattened z-stacks. Scale bar: 5 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 5. GFP-Mdv1p and -Caf4p partially localise to peroxisomes in a Fis1p-dependent manner. (A,D) caf4/mdv1/vps1 cells coexpressing GFP-Mdv1p (A) or GFP-Caf4p (D) and HcRed-PTS1 (per-Red). Top row: flattened z-stack showing faint colocalisation of GFP fusion with peroxisome (arrowheads). Bottom row: colocalisation is easier to detect in individual slices of the z-stack. (B,E) Mating experiment showing enhanced levels of Mdv1p and Caf4p on peroxisomes as a result of Fis1p-Pex15p expression. fis1/vps1 cells expressing both Pex15p-Fis1p and either GFP-Mdv1p (B) or GFP-Caf4p (E) were mated with pex3 cells expressing HcRed-PTS1. Top row: flattened z-stack showing colocalisation with peroxisomes. Bottom row: colocalisation is easier to detect in individual slices of the z-stack. Peroxisome labelling with GFP is much stronger compared to A and D. (C,F) Both GFP fusions were mainly cytosolic when expressed in fis1 cells. Scale bar: 5 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Peroxisome abundance in cells with mitochondrial dysfunction. (A) Cells expressing GFP-PTS1 were grown on 2% glucose medium to log phase and representative images were captured. Images are flattened z-stacks. Top rows, GFP-PTS1 fluorescence; bottom rows, phase-contrast images showing outline of cells. Scale bar: 5 µm. (B) Peroxisome numbers were counted from images of >100 budding cells for each strain.

View larger version (77K): [in this window] [in a new window]   Fig. 7. Dnm1p and Vps1p have distinct functions. Dnm1p overexpression does not restore the vacuolar fusion defect in vps1 cells, and Vps1p overexpression does not rescue the mitochondrial fission defect in dnm1 cells. (A) Vacuoles were visualised by allowing cells to take up FM4-64 to steady-state levels. The vacuolar membrane is clearly visible in wild-type (WT) cells and in vps1 cells expressing Vps1p, whereas unfused, fragmented vacuoles are visible in vps1 cells and in vps1 cells overexpressing Dnm1p. (B) Cells expressing mito-GFP were grown to log phase on 2% glucose. dnm1 cells overexpressing Vps1p show the mitochondrial fusion defect typical of dnm1 cells. Scale bar: 5 µm.