spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 2 January 2007
doi: 10.1242/jcs.03335

Journal of Cell Science 120, 340-352 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sigal, Y. J.
Right arrow Articles by Morris, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sigal, Y. J.
Right arrow Articles by Morris, A. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Cdc42 and ARP2/3-independent regulation of filopodia by an integral membrane lipid-phosphatase-related protein

Yury J. Sigal1, Omar A. Quintero2,*, Richard E. Cheney2 and Andrew J. Morris3,{ddagger}

1 Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, USA
2 Department of Molecular and Cellular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, USA
3 Division of Cardiovascular Medicine and Department of Molecular and Cellular Biochemistry, Gil Heart Institute, University of Kentucky College of Medicine, Lexington, KY 40536-6475, USA


Figure 1
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 1. LPR1 is a widely expressed integral membrane protein with a variant lipid phosphate phosphatase catalytic motif. (A) The deduced amino acid sequence of human LPR1 with hydrophobic residues that are predicted to form six transmembrane {alpha}-helices highlighted in yellow. Residues corresponding to the consensus phosphatase motif found on the LPPs and other family members are highlighted in red. The site of glycosylation is highlighted in purple and the sequence used to generate the LPR1 antibody is boxed. Residues shown in green font were mutated as described in the text. The C-terminus highlighted in green corresponds to the last 43 residues deleted to form the LPR1 C-term {Delta}43 mutant described in the text. The consensus phosphatase sequence motif is shown below in alignment with cognate sequences from four proteins of the LPR family. Residues that participate in the charge relay catalytic mechanism of the phosphatase reaction are highlighted in red and residues that contact the substrate or transition state phosphate group are highlighted in blue. Residues within this motif that are divergent in LPR1 and related proteins are underlined. (B) Membrane protein preparations from insect cells expressing LPP1 or LPR1 were analyzed by western blotting using an LPR1-specific antibody. Asterisk denotes an LPR1-specific immunoreactive band of 35-38 kDa. Samples contained equal quantities of proteins. (C) LPR1 expression in mouse tissues was analyzed by western blotting. Samples contained equal quantities of protein.

 

Figure 2
View larger version (85K):
[in this window]
[in a new window]

  		Fig. 2. LPR1 localizes to and increases the number of actin-rich membrane protrusions in HeLa and Cos7 cells. Cos7 cells were transfected with pEGFP, pEGFP-LPP3 (A) and pEGFP-LPR1 (B) and the distribution of EGFP-tagged protein and actin organization were visualized by immunofluorescence microscopy. (C) Bar graph comparing the number of filopodia, dorsal and peripheral, as visualized by phalloidin staining, in untransfected (control) Cos7 cells and Cos7 cells expressing either EGFP or EGFP-LPR1. Twenty cells were counted for each category; error bars denote s.d. for each group. (D). HeLa cells were transfected with pEGFP-LPR1 and localization of the protein and actin organization were determined by fluorescence microscopy. All images were acquired and processed identically. Panels B and D contain higher magnification images of sections of the cell periphery in the images above to show actin staining and LPR1 localization to filamentous structures. (E) Cell lysates from HeLa cells transfected with pEGFP, pEGFP-LPP3 or pEGFP-LPR1 were examined by western blotting with a GFP-specific antibody. Each lane was loaded with equal amounts of total protein. (F) Cos7 cells were transfected with pEGFP-LPR1, fixed, counterstained with Rhodamine phalloidin, and analyzed by confocal microscopy. Images are shown as projections of z sections in both Rhodamine and GFP channels. (G) Cos7 cells were transfected with pEGFP-LPR1, and examined live by confocal microscopy. The image represents a projection of z sections at a single time interval in a GFP-specific channel. Bars, 10 µm.

 

Figure 3
View larger version (85K):
[in this window]
[in a new window]

  		Fig. 3. LPR1-labeled filopodia are dynamic and exhibit both retractile and protrusive motion. Phase-contrast (lower frame) or EGFP fluorescence (upper frames) images of two HeLa cells expressing EGFP-tagged LPR1 are shown. The panels are individual frames taken from supplemental material Movies 1-4. The numbers on each panel denote the time at which each image was captured. (A) Arrows denote filopodia that steadily diminished in length throughout the period observed. (B) Arrows in each panel indicate either newly formed filopodia or filopodia that have increased in length compared with the previous time frame. Bars, 10 µm.

 

Figure 4
View larger version (98K):
[in this window]
[in a new window]

  		Fig. 4. Effects of LPR1 on filopodia are not mediated by the small GTPase cdc42. (A) HeLa cells were transfected with vectors for expression of myc-tagged cdc42Q 61L or cdc42T 17N. HeLa cells were transfected with vectors for expression of EGFP-LPR1 and myc-tagged constructs of cdc42T 17N (B), Wasp-CRIB (C) and Scar-WA (D). In all cases, EGFP- and myc-tagged proteins and actin organization were visualized by fluorescence microscopy. Bars, 10 µm.

 

Figure 5
View larger version (64K):
[in this window]
[in a new window]

  		Fig. 5. LPR1-induced filopodia resemble filopodia produced by expression of Rif, but LPR1-induced filopodia are not attenuated by co-expression with a dominant-negative-acting Rif mutant. (A) HeLa cells were co-transfected with plasmids expressing EGFP and either myc-tagged wild-type Rif (Rif WT myc), or myc-tagged constitutively active Rif (Rif QL myc). (B) HeLa cells were co-transfected with vectors encoding for myc-tagged dominant-negative Rif (Rif TN myc) and either EGFP alone or EGFP-tagged LPR1 (EGFP-LPR1). EGFP- and myc-tagged proteins, as well as actin, were examined by fluorescent microscopy in A and B. Bars, 10 µm. (C) HeLa cells were transfected with EGFP-LPR1 and either pcDNA, Rif TN myc or myc-Cdc42 (17N). EGFP-LPR1-labeled dorsal and peripheral filopodia were counted in 25 individual cells. The data shown are means ± s.d.

 

Figure 6
View larger version (91K):
[in this window]
[in a new window]

  		Fig. 6. LPR1-labeled filopodia have a unique protein composition. HeLa cells were transfected with pEGFP-LPR1, and immunofluorescence microscopy was used to visualize EGFP-tagged LPR1 and different endogenously expressed proteins: paxillin (A), Vasp (B) and myosin X (C). (D) Cos7 cells were transfected with vectors for expression of EGFP-LPR1 and localization of endogenous fascin and EGFP-LPR1 were analyzed by immunofluorescence microscopy. Fascin labels intracellular structures as well as membrane protrusions. High magnification images of the periphery of the cells show how these markers localize exclusively within the filopodia. Bars, 10 µm.

 

Figure 7
View larger version (60K):
[in this window]
[in a new window]

  		Fig. 7. LPR1 forms filopodia in the absence of Ena/Vasp proteins. MVD7 cells were transfected with vectors for expression of either EGFP or EGFP-LPR1 and visualized by fluorescent microscopy to analyze GFP fluorescence and either actin (A) or fascin (B). Bars, 10 µm.

 

Figure 8
View larger version (45K):
[in this window]
[in a new window]

  		Fig. 8. LPR1 is required for maintenance of membrane protrusions in ovarian epithelial cancer cells. (A) Proteins from SK-OV-3 cells that were either untransfected (control), or transfected with pEGFP alone or in combination with the control double-stranded RNA (RNAic) or two double-stranded siRNAs designed to target LPR1 (RNAi1 and RNAi2) were analyzed by western blotting for LPR1 expression. Immunoreactive species were quantified by scanning densitometry and pixel densities (normalized to control cells) are shown below each lane of the gel. (B) SK-OV-3 cells were co-transfected with pEGFP in combination with either RNAic or RNai2, and actin organization was visualized by fluorescence microscopy. Stars denote cells expressing the EGFP marker. Bars, 20 µm. (C) SK-OV-3 cells were transfected with EGFP alone or in combination with either RNAic or RNAi2. Phalloidin-stained filopodia were counted only in EGFP-expressing cells from the three different categories, and the averages of each were plotted. Error bars denote s.d. The average number of filopodia per cell in EGFP expressing cells was 56.7 (s.d. 20.6, n=21). EGFP-expressing cells co-transfected with RNAic had an average of 57.5 filopodia per cell (s.d. 13.5, n=26). EGFP expressing cells co-transfected with RNAi2 had an average number of 27.3 filopodia per cell (s.d. 12.3, n=25).

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2007