Zhang X et al. (2014), Overexpression of p49/STRAP alters cellular cyt...

XB-ART-49704

BMC Cell Biol 2014 Sep 02;15:32. doi: 10.1186/1471-2121-15-32.

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Overexpression of p49/STRAP alters cellular cytoskeletal structure and gross anatomy in mice.

Zhang X , Azhar G , Rogers SC , Foster SR , Luo S , Wei JY .

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BACKGROUND: The protein p49/STRAP (SRFBP1) is a transcription cofactor of serum response factor (SRF) which regulates cytoskeletal and muscle-specific genes. RESULTS: Two conserved domains were found in the p49/STRAP protein. The SRF-binding domain was at its N-terminus and was highly conserved among mammalian species, xenopus and zebrafish. A BUD22 domain was found at its C-terminus in three sequence databases. The BUD22 domain was conserved among mammalian p49/STRAP proteins, and yeast cellular morphogenesis proteins, which is involved in ribosome biogenesis that affects growth rate and cell size. The endogenous p49/SRAP protein was localized mainly in the nucleus but also widely distributed in the cytoplasm, and was in close proximity to the actin. Transfected GFP-p49/STRAP protein co-localized with nucleolin within the nucleolus. Overexpression of p49/STRAP reduced actin content in cultured cells and resulted in smaller cell size versus control cells. Increased expression of p49/STRAP in transgenic mice resulted in newborns with malformations, which included asymmetric abdominal and thoracic cavities, and substantial changes in cardiac morphology. p49/STRAP altered the expression of certain muscle-specific genes, including that of the SRF gene, which is a key regulator of cardiac genes at the developmental, structural and maintenance level and has two SRE binding sites. CONCLUSIONS: Since p49/STRAP is a co-factor of SRF, our data suggest that p49/STRAP likely regulates cell size and morphology through SRF target genes. The function of its BUD22 domain warrants further investigation. The observed increase in p49/STRAP expression during cellular aging may contribute to observed morphological changes in senescence.

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Species referenced: Xenopus
Genes referenced: actl6a eif3a nppa prim1 srf srfbp1 strap

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	Figure 1. Bioinformatic analysis of p49 protein sequence. A. A schematic of the p49 protein with two conserved regions. The SRF-binding domain is at the N-terminus; a BUD22 domain is located at the C-terminus. B. A BUD22 domain was found at the C-terminus of human, mouse and rat p49 proteins and three yeast BUD22 proteins, based on results from the Conserved Domain Database (NCBI), Pfam and InterPro databases. The lengths of the BUD22 domains in the six protein sequences illustrated in this diagram were based on the data from the Conserved Domain Database. According to the Conserved Domain Database, the BUD22 domain in human p49 was 29 amino acids in length. We found that this 29 amino-acid region (between two dash lines) was highly conserved in all six protein sequences. C. A schematic of the mouse p49 protein (Unipot ID Q9CZ91) which has four nuclear localization signal sequences (NLSs), and two nucleolar localization signal sequences (NoLSs). D. A schematic of the BUD22 protein of Baker’s yeast (Unipot ID Q04347), which has 3 NLSs and 3 NoLSs. E. A diagram of computational prediction of nucleolar localization signal sequence in mouse p49 protein (Unipot ID: Q9CZ91) using “NOD” (Nucleolar localization sequence Detector) website [44]. F. A diagram of computational prediction of nucleolar localization signal sequence in BUD22 of Baker’s yeast (Unipot ID Q04347) using “NOD” (Nucleolar localization sequence Detector) website [44].
	Figure 2. Intracellular distribution of p49/STRAP proteins. A-F: confocal microscopy revealed that p49 protein co-localized with nucleolin within the nucleolus in H9C2 cells. A. GFP protein is distributed in both cytoplasm and nucleus in GFP control plasmid transfected cells. B. Anti-nucleolin antibody was used to stain the nucleolus. Red fluorescence showed the nucleolus. C. Overlap of images of A and B. D. Transfected GFP-p49 protein was mainly distributed in nucleus and concentrated as dots within the nucleus. E. Anti-nucleolin antibody was used to stain nucleolin which is biomarker of nucleolus. Red fluorescence showed the nucleolin were stained as red dots. F. Overlap of images D and E, revealing GFP-p49 protein (in green) and nucleolin (in red) co-localized as yellow dots in the nucleolus. G-K: The distribution of endogenous p49 protein in H9C2 cells. The figures here revealed the abundance and the distribution of endogenous p49 protein in cells under normal culture condition. G. The endogenous p49 protein is widely distributed in the cell, but concentrated in the nucleus. H. Actin fiber was stained with rhodamine-phalloidin. I. Nuclei were stained with DAPI. J. Overlap of images A, B and C, which indicated that the p49 protein was in close proximity to the actin fiber. K. Digitally enhanced image showing that the p49 protein appeared to form a “tail” that was co-located at each end of the actin fibers, close to their attachment sites to the inner wall of the cell membrane.
	Figure 3. p49-adenovirus treatment reduced actin contents, and altered actin fiber structure as well as cell morphology. A-E: H9C2 cells that were infected with GFP-control adenovirus. A. Actin fibers were well expressed and well organized in control adenovirus infected cells. B. The green fluorescence revealed the nuclei which were stained with p49 antibody and an Alexa Fluor 488 labelled secondary antibody. C. Nuclei were stained with DAPI. D. Overlap of the image A, B and C. E. Digitally enhanced image showing control virus infection did not change actin contents and structure in H9C2 cells. There is fainter, less intensive visualization of the GFP protein in the cytoplasm surrounding the nucleus (Figure 3B, D and E). F-J: H9C2 cells that were infected with p49-adenovirus. F. Actin fiber signal intensity was reduced and actin fibers were disorganized. G. The green fluorescence revealed both endogenous and adenovirus delivered exogenous p49 proteins that were stained with p49 antibody and an Alexa Fluor 488 labelled secondary antibody. The p49/STRAP proteins were mainly localized in the nuclei. Part of the p49 protein was asymmetrically distributed within the cells (arrows). H. Nuclei were stained with DAPI. I. Overlap of the images F, G and H. The arrows pointed to the yellow “dots”, which were a result of the merger of green and red fluorescence, which indicated that part of the p49 proteins (in green) were co-localized with actin fiber (in red) within the cells. J. Digitally enhanced image showing that p49-adenovirus treatment reduced actin contents, and altered actin fiber structure as well as cell morphology.
	Figure 4. p49-adenovirus treatment reduced the actin fiber structure and intensity, and changed the cell morphology in term of cytoskeletal structure, cell size, cell shape and nucleocytoplasmic ratio. A. Control H9C2 cells were infected with GFP-control adenovirus and stained with Phalloidin and DAPI for visualization of F-actin and nuclei, respectively. Control cells showed relatively uniform and normal morphology for H9C2 cell line. Nuclei occupied a small, centrally located region of the cytoplasm. Actin fibers were dense and showed uniform network throughout the cytoplasm of the cell. B. P49/STRAP adenovirus infected H9C2 cells were stained with Phalloidin and DAPI for visualization of F-actin and nuclei, respectively. Overexpression of p49/STRAP in H9C2 cells showed smaller overall cell size, along with less uniform cellular shape compared to that of the control. Overall expression of actin was decreased, with significantly less uniformity among visualized fibers. Nuclei of p49/STRAP overexpressed cells were observed to occupy a much greater percentage of the cytoplasm and peripheral location in the cell. C. Histogram of cell size and numbers in H9C2 cell samples that were infected with p49-adenovirus versus control adenovirus. The cell size and number were measured under microscope and multiple fields were examined. The overall cell size in p49 adenovirus-infected samples was smaller versus control adenovirus-infected cells (n = 500, p <0.05). D. Histogram showed the nuclear to cytoplasmic ratio among control H9C2 cells infected with GFP only and adenovirus p49/STRAP infected cells. Data indicated that overexpression of p49/STRAP resulted in a significant increase in the nuclear to cytoplasmic ratio compared to that of the control (p < 0.05, n = 50). These results suggest that treatment with increased levels of p49/STRAP reduced cytoplasmic volume and overall cellular morphology of H9C2 cells.
	Figure 5. p49/STRAP repressed SRF and ANF gene expression. A. Effect of p49 expression on promoter activity of SRF gene. SRF gene promoter has two classic CArG box, therefore, SRF gene itself is regulated by SRF and SRF-cofactors. P49 had repressive effect on SRF gene promoter activity. B. p49 repressed the mRNA expression of SRF and ANF genes. * refers to p < 0.05, n = 3; ** refers to p < 0.01, n = 3.
	Figure 6. Morphological change in p49Tg mice versus non-Tg mice. A. Image of p49Tg newborn (Tg) versus Non-Tg newborn. The arrow indicates the p49Tg newborn missed a leg. B. The arrows highlight the contracture deformities of the limbs with additional webbing or non-delineated appendages in the p49Tg newborn. The tissue sections from C to H were stained with H&E staining. C and D. Longitudinal section of p49Tg newborn versus non-Tg newborn, which revealed that the spine in the M-p49Tg newborn was shifted around to the abdominal cavity from the back to the front (dorsal to ventral). E. Cross sections of non-Tg newborn. F. Cross section of Tg newborn with asymmetric thoracic cavity. Both the left and right lungs were unexpanded in the dead newborn the Tg mouse. G. Tissue section of non-Tg heart. H. Tissue section of p49Tg heart, which revealed malformed heart.
	Figure 7. Gene expression of p49 and other genes in p49Tg mouse tissue and cells versus that of non-transgenic mice. A. Gene expression in malformed p49Tg newborns versus NTg newborns. The data indicated that p49 overexpression altered the expression of cytoskeletal genes. * refers to p < 0.05, n = 3; ** refers to p < 0.01, n = 3. B. Aortic smooth muscle cells (isolated from 3 month old Tg and NTg) were treated with sodium butyrate for 24 hours. P49 expression was not significant between Tg cells and NTg cells in the absence of sodium butyrate (No treatment). There was slight increase in p49 expression Tg cells in the presence of 0.5 mM sodium butyrate, but not significant (p = 0.054, n = 3). p49 mRNA was significantly increased in the presence of 5 mM sodium butyrate (p < 0.01, n = 3).

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Ahmad, NOPdb: Nucleolar Proteome Database--2008 update. 2009, Pubmed