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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02472

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Dynein light chain rp3 acts as a nuclear matrix-associated transcriptional modulator in a dynein-independent pathway

¹ Department of Ophthalmology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA
² Department of Cell and Development Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA

View larger version (88K): [in a new window]   Fig. 1. Subcellular distribution of rp3. (A) Confocal images of HeLa cells double labeled for rp3 and IC. rp3 appears at the centrosome (open arrow, left) and microtubule plus-end, which often colocalized with IC in the cytoplasm (arrows, middle). The focal plane was set so that both cytoplasmic and nuclear signals of rp3 are shown in a single image. In the nucleus, rp3 appears on punctate structures that were occasionally associated with IC labeling (arrowheads, right). Nuclei are circled. (B) Proteins from cytosolic and nuclear fractions of HeLa, HEK and dissected brain regions were analyzed by immunoblotting with the indicated antibodies. (C) Immunolabeling of endogenous rp3 and PML in paraformaldehyde-fixed and Triton X-100-extracted HeLa cells (top panels) or in Triton X-100-extracted, paraformaldehyde-fixed and DNaseI-digested HeLa cells (bottom panels). Lack of DAPI signal in the bottom row indicates complete DNA removal. Bar, 5 µm.

View larger version (57K): [in a new window]   Fig. 2. Interaction between SATB1 and rp3. (A) Recombinant GST, GST-rp3 and GST-Tctex-1 immobilized on glutathione beads were incubated with in vitro-translated, 35S-labeled Flag-SATB1. Eluted proteins were either Coomassie Blue stained (top), or transferred for autoradiography (bottom). Roughly equivalent amounts of GST fusions were used in each experiment (arrows). (B) Anti-Flag antibody immunoprecipitates obtained from HEK cells overexpressing the indicated proteins were immunoblotted with the indicated antibodies (top three rows). The bottom three rows represented the total input used in the immunoprecipitation experiments. Note that a small amount of endogenous Tctex-1 signal was also detected in non-transfected cells using anti-Tctex-1 antibody (bottom row).

View larger version (87K): [in a new window]   Fig. 3. Nuclear localization of rp3, facilitated by its interaction with the N-terminal region of SATB1. (A) Schematic diagram of SATB1 and the expression constructs of SATB1 used for transfection (top). Boxes label the PDZ domain, the MAR binding domain and the atypical homeodomain (HOX) motif. Immunolabeling of Flag-tagged SATB1, SATB1-N and SATB1-C in transfected HEK cells (bottom). (B) HEK cells co-transfected with rp3 and various Flag-SATB1 constructs were immunoprecipitated with anti-Flag antibody followed by immunoblotting for either Flag or rp3. The immunoblots of total inputs are also shown. (C) Colocalization study of HEKs singly transfected with GFP-rp3, or double transfected with GFP-rp3 or GFP-IC, together with various Flag-SATB1 constructs for GFP (green) and Flag (red). (D) Nuclear Flag-SATB1 (red) and GFP-rp3 (green) were co-distributed in the interchromatin spaces surrounding heterochromatin, the dense regions of DAPI staining, in transfected cells (top panel). Nuclear colocalized Flag-SATB1 and GFP-rp3 remained in the nuclear matrix after DNaseI digestion (bottom panel). Bar, 5 µm.

View larger version (82K): [in a new window]   Fig. 4. Nuclear transport of SATB1 and rp3. (A) HEK cells triple transfected with pTet-off, TRE-Flag-SATB1 and myc-p50 were cultured in the presence of doxycycline for 24 hours and switched to regular medium for an additional 24 hours prior to fixation. The fixed cells were double-labeled with anti-Flag and anti-myc (top) or anti--adaptin and anti-myc antibodies (bottom). The centrosomal Golgi distributions in nontransfected cells are indicated (arrowheads). Dotted outlines represent cell margins. (B) Immunoblotting of total cell lysate extracts obtained from cells transfected with either rp3 siRNA or control siRNA and probed with anti-ß-tubulin and anti-rp3. (C) HEK cells were triple transfected with pTet-off, TRE-Flag-SATB1, together with either rp3 siRNA or control siRNA. The Flag-SATB1 expression was manipulated as described in A and the cells were immunolabeled with anti-Flag mAb. Bar, 5 µm.

View larger version (59K): [in a new window]   Fig. 5. SATB1 association with rp3 free from dynein complex and IC. (A) Cytosolic and nuclear extracts of HEK cells sedimented in a 5-20% linear sucrose gradient. Fractions were collected, analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. Each nuclear fraction was immunoprecipitated (IP) with anti-rp3 or control rabbit IgG and immunoblotted (IB) with anti-SATB1 antibody (bottom panel). (B) HEK nuclear extracts were immunoprecipitated with either anti-IC or anti-rp3 and immunoblotted with the indicated antibodies. Immunoblots of total inputs are also shown. (C) Nuclear extracts underwent sequential immunoprecipitation procedures. The immunoprecipitates were subjected to immunoblotting with the indicated antibodies. Location of standards for 3.5S, 11.3S and 19S are indicated.

View larger version (75K): [in a new window]   Fig. 6. rp3 assembly within the SATB-1-Bcl2 MAR DNA complexes. Radiolabeled Bcl2 MAR was incubated with nuclear extracts of HEK cells transfected with SATB1 (lanes 1-5) or SATB1 together with Flag-rp3 (lanes 6-14). The indicated antibodies were added into each reaction. IC-depleted nuclear extracts were used in lanes 11-14. Arrows indicate the SATB1-containing Bcl2 MAR complexes (I/II). Asterisks label the slower migrating complexes generated by anti-SATB1 (lanes 2, 7, 12) and anti-Flag antibodies (lanes 8, 13).

View larger version (48K): [in a new window]   Fig. 7. rp3 involvement in SATB1-mediated transcriptional repression activity and gene regulation of Bcl2. (A) HEK cells were transfected with pGL2-5Gal, pTK-RL plus the indicated plasmids. The luciferase activity was measured and that of pcDNA3.1-transfected cells was taken to be 100% (column 5). (B) Luciferase assays of HEK cells transfected with pTK-RL, pGL2-5Gal, pGal4DB-SATB1 and rp3 siRNA or control siRNA. The luciferase activity obtained from cells transfected with pcDNA3.1 and treated with control siRNA was taken to be 100%. (C) rp3 reversed SATB1 suppression effect on Bcl2-MAR. Luciferase assays were performed using lysates of HEK cells transfected with pTK-RL, Bcl2-MAR-Luc and plasmid combination as indicated. The activity of cells transfected with pRK5 was taken as 100%. (D) Bcl2 mRNA levels were measured by real-time RT-PCR using RNAs isolated 24 hours after transfection with the indicated plasmids. The Bcl2 mRNA level of pRK5-transfected cells was taken to be 100%. Significant differences in luciferase activity and mRNA levels were observed (**P<0.01) in indicated cells compared to levels in the control. All values represent the mean±s.e.m. of three separate experiments.

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