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First published online 9 August 2005
doi: 10.1242/jcs.02516

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Extra-mitochondrial localisation of frataxin and its association with IscU1 during enterocyte-like differentiation of the human colon adenocarcinoma cell line Caco-2

¹ Dipartimento di Biologia e Patologia Cellulare e Molecolare, Via S. Pansini 5, Istituto di Endocrinologia ed Oncologia Sperimentale Centro Nazionale delle Ricerche, Università `Federico II', Napoli, Italy
² Unità Operativa Complessa di Anatomia Patologica, Istituto Nazionale Tumori, Via M. Semmola, 80131 Napoli, Italy
³ Unità Operativa Complessa di Oncologia Medica B, Istituto Nazionale Tumori, Via M. Semmola, 80131 Napoli, Italy

View larger version (19K): [in a new window]   Fig. 1. (A) Western blot analysis of frataxin and VDAC-1 expression in Caco-2 cells extracts at different stages of culture. (B) Relative densitometric analysis of mature frataxin and VDAC-1 expression was normalised to tubulin using NIH Image Software. Data is expressed as fold change over control levels at day 4. (C) D-loop and Cytochrome-b mtDNA expression analysis in Caco-2 cell after the indicated days of culture. Results are expressed as fold change over control levels at day 4. Experiments were carried out three times; results presented are taken from one representative experiment.

View larger version (11K): [in a new window]   Fig. 2. Quantitative gene expression of frataxin using real-time PCR in Caco-2 cells at the indicated days of culture. Fold changes (y-axis) represent the relative expression arbitrarily setting the mRNA frataxin level at day 4 to 1. Indicated values, normalised to ß-glucuronidase mRNA level, are means ±s.d. of three independent experiments.

View larger version (157K): [in a new window]   Fig. 3. Immunohistochemical staining of human intestinal mucosa. The staining for frataxin was performed using anti-frataxin antibody. Along the crypt-villus axis the stem cells committed to become mature enterocytes migrate and differentiate. Proliferating undifferentiated cells are located towards the base of the crypt compartment, while differentiated cells are located at the tips of the villus. Frataxin signal corresponding to the brown staining become clearly evident in the upper half of the villus (arrowheads indicate). Magnification used is 40x.

View larger version (91K): [in a new window]   Fig. 4. Double-immunostaining and confocal analysis of permeabilized Caco-2 cells with anti-frataxin antibody. (A) In proliferating cells at day 4 of culture, frataxin localises in elongated cytoplasmic organelles identified as mitochondria by MitoTracker Red staining. (B) Differentiated cells (day 13) were smaller in size and contained a small amount of cytoplasm. Frataxin staining appears diffuse in the cytosol and co-localises only in part with MitoTracker Red signal. The arrows indicate a site of cell-cell contact clearly positive for frataxin. Magnification used is 100x.

View larger version (66K): [in a new window]   Fig. 5. Subcellular localisation of frataxin, VDAC-1 and citrate synthase in proliferating and differentiated Caco-2 cells. The enrichment of mitochondria in membrane/organelle fractions was checked by the use of VDAC-1 and citrate synthase mitochondrial markers. Membrane/organelle-enriched fractions contain mitochondria (M.F.), while the cytoplasmic fraction (C.F.) does not.

View larger version (24K): [in a new window]   Fig. 6. Fluorescence activated cell-sorting analysis of Mn-SOD and frataxin expression in permeabilized and non-permeabilized Caco-2 cells. The red lines indicate the non-specific anti-mouse antibody fluorescence, while the blue and green lines indicate Mn-SOD and frataxin specific fluorescence respectively in proliferating and differentiated Caco-2 cells. (A) Non-permeabilized Caco-2 cells. Frataxin-specific cell-surface fluorescence is evident at day 13. (B) Permeabilized Caco-2 cells. (C) Percentage of surface-frataxin positive cells; analysis of one of three independent experiments.

View larger version (48K): [in a new window]   Fig. 7. Western blot analysis of IscU1 and mitochondrial marker VDAC-1 expression during Caco-2 cell differentiation. IscU1 and VDAC-1 protein levels from total, cytosolic (C.F.) and mitochondrial (M.F.) fractions of Caco-2 cells at day 4 and 13 of culture are shown.

View larger version (32K): [in a new window]   Fig. 8. Cytosolic association of IscU1 with frataxin during Caco-2 cell differentiation. Western blot analysis of IscU1 and frataxin protein levels on total cell extract and on cytosolic fraction (C.F.) at day 4 and 13 of culture following immunoprecipitation with anti IscU1 or anti-frataxin antibody.