Particle-tracking analysis of GFP-SKL-marked peroxisomes in CHO cells.

<p>(A) Treatment of cells with <i>C. botulinum</i> toxin exoenzyme C3 (5 mg/ml, 24 h prior to analysis) known to inactivate RhoA significantly increases the number of peroxisomes moving long distances. (B) In cells expressing dominant-active V14-RhoA long-range movement is abolished and peroxisomal motility is indistinguishable to that in cells treated with nocodazole to depolymerize microtubules. Expression of dominant-active V26-RhoD does not show this effect. For clarity reasons the displacement graph of control peroxisomes expressing empty vector is omitted, as it is virtually identical to the control graph in (A).</p

ATP- and GTP-dependent binding of NMM IIA heavy chain and b-actin to isolated rat liver peroxisomes.

<p>Organelles were incubated as indicated and floated up in a Nycodenz gradient. Three fractions were recovered from top to bottom (F1–F3). Binding is visualized by immunoblotting (A and C). ATP must be hydrolyzable for binding (E). Peroxisomes are localized in the gradient by the peroxisomal marker Pex11ap. Control incubations lacking peroxisomes do not show floatation of NMM IIA and b-actin (B, D, F) suggesting peroxisomal membranes to be required for binding.</p

Intracellular localization of Rab8a.

<p>Huh7 cells were stably transfected with dominant-active (GFP-Rab8aQ67L; A and J), dominant-inactive (GFP-Rab8aT22N; D and K) and wild type Rab8a (GFP-Rab8aWT; G) constructs. Peroxisomes were visualized by using primary anti-catalase (B, E and H) and Golgi cisternae were stained with primary anti-GM130 (J and K). Alexa FLUOR-labeled secondary antibodies were used for staining. Images were assembled from z-projections. The bar represents 10 mm. The quantitative evaluation of peroxisomes co-localizing with Rab8a following transfection of wild type and mutant Rab8a is shown in (L). n =  number of cells evaluated.</p

Co-localization of RhoA and peroxisomes in AT3 mouse hepatoma cells.

<p>Endogenous (A, C) and transfected dominant-active myc-G14V-RhoA (D, F, red) co-localize to peroxisomes (C, F) visualized by the peroxisomal matrix protein catalase (B, E, green). At higher magnification (inset in F) a patchy distribution of RhoA on peroxisomes becomes apparent. Inactivation of RhoA by <i>C. botulinum</i> exoenzyme C3 abolishes RhoA localization to peroxisomes (G–I). Images were reproduced from single z-layers. The bars represent 10 mm.</p

GTP-dependent recruitment of RhoA onto isolated rat liver peroxisomes.

<p>No recruitment is seen in the presence of ATP alone (A) and no RhoA is floating up, if peroxisomes are omitted from the incubations (B). Note that without membranes more RhoA precipitates during incubation escaping analysis.</p

Localization of peroxisomes to acto-myosin complexes in wild type CHO cells.

<p>Most peroxisomes visualized by the membrane marker PMP69 (red) are found attached to NMM IIA-associated acto-myosin filaments (A–C). In many cases a number of peroxisomes is aligned along single filaments (see inset) suggesting that peroxisome distribution within the cell is not at random but may be determined by the organelle′s association to acto-myosin filaments (D–F). Images are reproduced from single z-layers. The bar represents 10 mm.</p

Cytoskeleton and cytoskeleton-related proteins associated with peroxisomes, identified by nano-HPLC/ESI-MS/MS.

<p>Cytoskeleton and cytoskeleton-related proteins associated with peroxisomes, identified by nano-HPLC/ESI-MS/MS.</p

Recruitment of ROCKII onto isolated rat liver peroxisomes.

<p>Isolated organelles already have bound small amounts of ROCKII (lane 6 in A). In the presence of ATP and particularly ATP plus GMP-PNP this amount is increased about 2-fold and 5-fold, respectively (lanes 1 and 3 in C). Localization of peroxisomes in the gradient following floatation is indicated by the peroxisomal membrane marker PMP69 (A). Omitting peroxisomes from the incubations abolishes ROCKII floatation (B).</p

Ultrastructural analysis of peroxisome proliferation in rat liver treated with clofibrate and thyroxin.

<p>Peroxisomal constrictions are frequently seen in peroxisomes (P) of different sizes (black arrows in A and F) as well as in peroxisomes associated to microtubular tracks (black arrowheads in B) that sometimes localize close to the constrictions (black arrowhead in C). Filament bundles are running along peroxisomes and close to peroxisomal constrictions (white arrows in D–F) suggesting peroxisome attachment to cytoskeletal filaments. Frequently, cisternae of smooth endoplasmic reticulum (white arrowheads in A–C) are positioned next to the constrictions. M, mitochondrium. The bars represent 200 nm in A, B, D–F and 100 nm in C.</p