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Interaction of mitochondria with microtubules in the filamentous fungus Neurospora crassa

Institut für Physiologische Chemie der Universität München, Butenandtstr. 5, D-81377 München, Germany

View larger version (18K): [in a new window]   Fig. 1. Plasmid for expression of mitochondria-targeted GFP in Neurospora crassa. (A) Structure of the mtGFP cassette. The sequence coding for GFP including a stop codon is symbolised by a white box, regulatory sequences of the atp-1 gene are depicted in grey, introns are delineated by black lines, and regions coding for parts of the F1 protein are depicted by black boxes. F1 pre, region of the atp-1 gene coding for the mitochondrial presequence; F1C, region of the atp-1 gene coding for the C-terminus of the F1 protein (not translated in the chimeric construct); atp-1 ter., atp-1 terminator. (B) Plasmid map for pNc-mtGFP. All relevant functional elements are indicated. The ampicillin resistance gene (ampr) and the origin of replication for E. coli (ColE1 ori) are derived from the pBluescript vector. The plasmid map is not drawn to scale. Unique restriction sites are underlined. See text for cloning details.

View larger version (70K): [in a new window]   Fig. 2. GFP-labelled mitochondria in living Neurospora cells. Conidiospores of a strain expressing mtGFP were grown for different time periods in Vogel's liquid minimal medium at 30°C under agitation and subjected to fluorescence (left) and phase contrast (right) microscopy. (A) Conidiospore before germination; (B) newly germinated conidiospore; (C) hyphal tip after 4 hours incubation; (D) hypha after overnight incubation; (E) hyphal branch point after overnight incubation. Bar, 5 µm.

View larger version (88K): [in a new window]   Fig. 3. Behaviour of isolated mitochondria. (A) Mitochondria were isolated from a Neurospora strain expressing mtGFP and subjected to fluorescence (left) and phase contrast (right) microscopy. The insert shows an enlarged section from the center of the image. (B) Isolated mtGFP-containing mitochondria were incubated with rhodamine-labelled microtubuli and subjected to fluorescence microscopy. Bars, 5 µm (A,B); 1 µm (A,inset).

View larger version (27K): [in a new window]   Fig. 4. Binding of microtubules to mitochondria in vitro. (A) Isolated mitochondria were incubated with and without an excess of microtubules and subjected to floatation centrifugation in a sucrose density gradient. After harvesting fractions of the gradient were precipitated with TCA and analysed by SDS-PAGE and Coomassie staining. Tubulin floated with mitochondria is marked with an asterisk. (B) Isolated mitochondria were incubated with microtubules, and floated in a sucrose density gradient; fractions were harvested, proteins were precipitated and analysed by SDS-PAGE and western blot. Only fractions 1 (containing floated mitochondria as controlled by blotting against porin), 3 (from the middle of the gradient) and 5 (containing non-floated proteins) are shown. P, pellet from the bottom of the gradient. Top panel, standard: mitochondria incubated with microtubules under standard conditions (i.e. in the absence of adenine nucleotides and without pretreatment of organelles). Panels 2 and 3: trypsin-pretreated mitochondria and salt-washed mitochondria, respectively, which were incubated with microtubules under standard conditions. Panels 4 and 5: non-pretreated mitochondria that were incubated with microtubules in the presence of ATP or ADP, respectively.

View larger version (29K): [in a new window]   Fig. 5. Binding of microtubules to mitochondria lacking MMM1. Mitochondria isolated from a mmm-1RIP mutant were incubated with microtubules under standard conditions or in the presence of ATP and analysed as described for Fig. 4B.

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