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First published online November 10, 2004
doi: 10.1242/10.1242/jcs.01502

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Inducible changes in cell size and attachment area due to expression of a mutant SWI/SNF chromatin remodeling enzyme

David A. Hill^1,*, Simion Chiosea^1,*, Saha Jamaluddin³, Kanaklata Roy¹, Andrew H. Fischer², Douglas D. Boyd³, Jeffrey A. Nickerson^1, and Anthony N. Imbalzano^1,

¹ Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA
² Department of Pathology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA
³ Department of Cancer Biology, The University of Texas-MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA

View larger version (57K): [in a new window]   Fig. 1. Changes in cell physical appearance but not in the rate of cell division upon expression of mutant BRG1. (a) Cells expressing mutant BRG1 (–tet) change appearance and reach confluence faster than control cells (+tet). (b) Cell counts for each cell line grown in the presence or absence of tetracycline for 3 days.

View larger version (75K): [in a new window]   Fig. 2. Mutant BRG1 causes an increase in the size of the cell and the nucleus. (a) Image of a CSFE-labeled cell (left) and the corresponding DIC image (right). Bar, 10 µm. (b) Distribution of cell surface attachment area (in µm2) for B05-1 cells grown in the presence (n=65) or absence (n=61) of tetracycline for 3 days. Cells were at 20-25% confluence to permit examination of individual cells. (c) Distribution of cell volume in (in µm3) for B05-1 cells grown to 20-25% confluence in the presence (n=60) or absence (n=56) of tetracycline for 3 days. (d,e) B05-1 cells grown in the presence (d) or (e) absence of tetracycline for 3 days were Papanicolaou stained to examine the appearance and size of the nucleus. Bar, 10 µm.

View larger version (42K): [in a new window]   Fig. 3. Expression of mutant BRG1 causes a change in the size and distribution of focal adhesions but not in paxillin or actin protein levels. Immunostaining for (a,b) paxillin, (c,d) phalloidin, (e,f) the merged images of a,c and b,d, respectively, and (g,h) DAPI staining in B05-1 cells grown to approximately 50% confluence in the presence (a,c,e,g) or absence (b,d,f,h) of tetracycline for 3 days. Bar, 10 µm. (i) Western blot demonstrates that total cellular paxillin and actin levels are unchanged by tetracycline or expression of mutant BRG1 in three different cell lines. Flag immunoreactivity demonstrates that the mutant BRG1 was expressed.

View larger version (53K): [in a new window]   Fig. 4. Cells expressing mutant BRG1 show increased levels of some adhesion proteins. Western blots against urokinase receptor (a) and V and 5 integrins (b) indicate increased levels in each of the three cell lines expressing mutant BRG1. Phosphatidylinositol 3-kinase (PI3K) levels were monitored as a loading control; Flag levels indicate the level of mutant BRG1 protein expressed in each line. The tet-VP16 samples probed for uPAR levels were run on a separate gel and spliced into the figure.

View larger version (13K): [in a new window]   Fig. 5. Expression of ATPase-deficient BRG1 significantly reduces the invasion of Matrigel membrane by B05-1 cells (P<0.05, by Mann-Whitney non-parametric test). The average number of invading cells is presented ±s.d.

View larger version (55K): [in a new window]   Fig. 6. uPAR mRNA levels are not misregulated by dominant negative BRG1. Northern blot analysis shows levels of uPAR and ß-actin mRNAs in two different lines (B05-1 and B22) that express dominant negative BRG1 upon removal of tetracycline.