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 11 July 2006
doi: 10.1242/jcs.03026

Journal of Cell Science 119, 3098-3106 (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 Related articles in JCS
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 Schauss, A. C.
Right arrow Articles by Jakobs, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schauss, A. C.
Right arrow Articles by Jakobs, S.
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?

Fis1p and Caf4p, but not Mdv1p, determine the polar localization of Dnm1p clusters on the mitochondrial surface

Astrid C. Schauss, Jörg Bewersdorf* and Stefan Jakobs{ddagger}

Max-Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Am Fassberg 11, 37077 Göttingen, Germany


Figure 1
View larger version (61K):

[in a new window]
  		Fig. 1. Deletion of FIS1, CAF4 or MDV1 influence the relative sizes of the mitochondria-associated Dnm1p-GFP pools. The histogram displays the relative amounts of mitochondria-associated Dnm1p-GFP analyzed on a single-cell basis. 100% equals the whole cellular Dnm1p-GFP pool of the respective cell. For the analysis, fixed cells expressing Dnm1p-GFP and mtDsRed were imaged by using single-molecule-sensitive quantitative confocal fluorescence microscopy. The obtained image data sets were semi-automatically scrutinized. For each strain 80 cells were analyzed.

 

Figure 2
View larger version (27K):

[in a new window]
  		Fig. 2. Cytosolic and mitochondria-associated Dnm1p-GFP assemblies have different size distributions in wt, mdv1{Delta}, fis1{Delta} and caf4{Delta} cells. (A) Fluorescence intensity distribution of GFP-VLPs normalized to 1. The major peak corresponds to single GFP-VLPs, whereas the second peak corresponds to two aggregated GFP-VLPs. A single GFP-VLP contains 120 GFP molecules. (B-E) Fluorescence intensity distributions of mitochondria-associated (gray) and cytosolic (black) Dnm1p-GFP assemblies normalized to the mean fluorescence intensity of single GFP-VLPs. For each graph 80 cells expressing Dnm1p-GFP and mtDsRed were imaged and analyzed. Note the differing y-axes values in (E). Insets show representative fluorescence images. Bar, 1 µm.

 

Figure 3
View larger version (37K):

[in a new window]
  		Fig. 3. Tubule constrictions are encircled by a Dnm1p spiral (or rings) that usually contain 100 to 500 Dnm1p molecules. (A-C) Confocal image of a wt cell expressing mtDsRed (A), Dnm1p-GFP (B) and the overlay of both images together with a bright field image (C). The arrow points to a tubule constriction coinciding with a Dnm1p-GFP assembly. Bar 1 µm. (D) Fluorescence intensity profiles in the GFP and DsRed channel (added up in the y-direction) within the rectangle shown in C at a tubule constriction. (E,F) Fluorescence intensity distributions of GFP-VLPs (gray) and Dnm1p-GFP spirals (green) normalized to the average fluorescence intensity of a single GFP-VLP in wt (E) and caf4{Delta} cells (F). Note the differing y-axes values in E and F. All Dnm1p-GFP spiral-like structures were scrutinized manually. One GFP-VLP contains 120 GFP molecules.

 

Figure 4
View larger version (32K):

[in a new window]
  		Fig. 4. Fis1p and Caf4p, but not Mdv1p, are required for the polar localization of Dnm1p-GFP clusters on mitochondrial tubules. (A) In wt, mdv1{Delta}, fis1{Delta}, and caf4{Delta} cells expressing Dnm1p-GFP and mtDsRed the precise localizations of mitochondria-associated Dnm1p-GFP clusters were analyzed. The polarity was observed in live and chemically fixed wt cells. The deletion mutants were fixed prior to analysis to avoid spatial movements of the clusters. For each strain more than 80 cells were evaluated. The Dnm1p-GFP clusters point to the cell center (in) or to the cell cortex (out). Due to the curvature of the cell surface, the employed algorithm calculates for randomly distributed mitochondria-associated clusters a slight bias in the distribution (calc.). (B) Top: Bright-field image of a wt cell expressing Dnm1p-GFP and mtDsRed. Bottom: Add-up of confocal sections showing a mitochondrial Dnm1p-GFP cluster pointing to the cell cortex (1), a cluster with a non-analyzable (sideward) localization (2), a mitochondrial Dnm1p-GFP cluster pointing to the cell center (3) and a cytosolic Dnm1p-GFP assembly (4). Only clusters of the categories 1 (out) and 3 (in) were included in the analysis. Bar, 1 µm.

 

Figure 5
View larger version (58K):

[in a new window]
  		Fig. 5. Polar localization of mitochondrial Dnm1p-GFP clusters is maintained in the absence of F-actin. (A) The actin cytoskeleton partly colocalizes with the mitochondrial network in wt cells. (Left panel) Actin cytoskeleton stained with phalloidin-conjugated Alexa Fluor-546. (Middle panel) GFP-labelled mitochondria. (Right panel) Corresponding bright-field image. (B) Bar graphs show the polarity of the mitochondria-associated Dnm1p-GFP clusters in wt cells treated with the actin depolymerizing drug Latrunculin A for 40 minutes (LatA 40') or without (untreated). A majority of the clusters point to the cell cortex (out). Right: Bright-field and fluorescence image of a representative wt cell treated with Latrunculin A for 40 minutes expressing Dnm1p-GFP and mtDsRed. Bars, 1 µm.

 

Figure 6
View larger version (86K):

[in a new window]
  		Fig. 6. Polarized mitochondria-associated Dnm1p-GFP clusters appear to be physically attached to the cell cortex. Shown is a wt cell expressing Dnm1p-GFP and mtDsRed after formaldehyde fixation. Displayed is an add-up of confocal sections of the two fluorescence channels overlaid with a bright-field image. After formaldehyde fixation the mitochondrial tubules retract occasionally from the cell cortex, as shown in this image. Arrowheads point to mitochondria-associated Dnm1p-GFP clusters that appear to be attached to the cell cortex. The arrow points to a Dnm1p-GFP cluster that stayed at the cell cortex and might have been torn off from the mitochondrial tubule. Bar, 1 µm.

 

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