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First published online 23 January 2007
doi: 10.1242/jcs.03364

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Rac1 and Rac3 have opposing functions in cell adhesion and differentiation of neuronal cells

Amra Hajdo-Milainovi, Saskia I. J. Ellenbroek, Saskia van Es, Babet van der Vaart and John G. Collard^*

The Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

View larger version (37K): [in this window] [in a new window]   Fig. 1. Both Rac1 and Rac3 are endogenously expressed in N1E-115 cells, and are efficiently and specifically downregulated by shRNA. (A) Total RNA isolated from N1E-115 was used as template for cDNA synthesis. Primers specific for Rac1, Rac3 or GAPDH were used in semi-quantitative reverse-transcription PCR to show the relative expression levels of these genes. (B) Cells were transfected with pcDNA3.1 empty vector (ev), Rac3FLAG or Rac1HA, lysed and western blotted. Upper panel shows total Rac staining. Note that Rac3 antibody recognizes specifically Rac3 and not Rac1 (middle panel). -actin staining was used as loading control. (C) RT-PCR showing the expression levels of GAPDH (control; top panel), endogenous Rac3 (middle panel) and endogenous Rac1 (lower panel) in cells in which either shRac1 (lane 2, 3) or shRac3 (lane 4, 5) has been expressed. Control cells (lane 1) were transfected with siLuc. (D) Western blot showing depletion of Rac3 protein upon expression of shRac3, visualized by total Rac antibody (upper panel) and Rac3-specific antibody (middle panel). Beta actin staining is shown as loading control. (E) Western blot showing depletion of either Rac1 or Rac3 by shRac1 or shRac3, respectively. pSuper constructs, containing different sequences specifically targeting Rac1, Rac3 (e.g. shRac1-1, shRac3-1 and shRac3-2), or luciferase, were co-transfected with pcDNA3/Rac1HA or pcDNA3/Rac3FLAG constructs in HEK293 cells.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Rac1 and Rac3 produce opposite effects on N1E-115 morphology. (A) Cells were transfected with pcDNA3/Rac1HA, pcDNA3/Rac3FLAG, pSuper/shRac1 or pSuper/shRac3, together with a green fluorescent protein vector (ratio 10:1). Subsequently, cells were serum-starved for 24 hours, fixed and stained with phalloidin to visualize filamentous actin. Bar, 25 µm. (B) Quantification of the changes in morphology as seen in A from at least three independent experiments (50-100 cells counted per experiment). Cells were scored as flat, round or protrusion bearing. P values: protrusions in Rac1 versus control *P=0.2; rounded cells in Rac33 vs control **P=0.014; rounded cells siRac1 vs control ***P=0.038; protrusion outgrowth in siRac3 vs control ****P=0.003. (C) Cells were transfected with pcDNA3.1 empty vector (ev), mouse-specific shRac3 together with ev, or mouse-specific shRac3 combined with human Rac3 cDNA. Protrusion-bearing cells (where protrusion is >1x cell body) were counted and depicted in a bar graph. Note that the phenotype induced by mouse-specific shRac3 is prevented by co-expression of human Rac3. (D) Rac3 is downregulated in differentiated serum-starved N1E-115 cells. Serum-cultured and serum-starved N1E-115 cells were analyzed by RT-PCR for the amount of Rac1 and Rac3 mRNA. (E) Rac3 depletion induces differentiation of N1E-115 cells as shown by various differentiation markers. Parental N1E-115 cells were either transfected with shRac3 (together with eGFP in a ratio of 10:1, to track the transfected cells) and cultured under normal conditions (right panels) or differentiated by 24 hours serum-starvation and 4 hours NGF treatment (left panels). The cells were stained with either anti-neurofilament 200 (NF200) antibody (upper panels) or anti-microtubule-associated protein 2 antibody (lower panels).

View larger version (46K): [in this window] [in a new window]   Fig. 3. Specific Rac3-induced cell rounding and intracellular localization are dependent on amino acid residues 185-187. (A) Schematic representation of chimeric Rac3/Rac1 mutants used in the experiments. The asterisks indicate divergent residues. (B) Transient expression of Rac3*CAAX1 and Rac3*Cterm1 mutants in N1E-115 cells. eGFP-containing construct was co-transfected in a 1:5 ratio, to visualize transfected cells. 24 hours after transfection, cells were serum-starved for 24 hours, stained with phalloidin to visualize F-actin and photographed. Bar, 25 µm. Note that Rac3*CAAX1-induced morphology is similar to the cell rounding induced by the parental Rac3 protein, whereas the Rac3*Cterm1 mutant induces cell relaxation and spreading similar to the effect of Rac1. (C) Morphologies observed in B were scored as flat, round or protrusion bearing in two independent experiments, and presented in a bar-diagram. Control cells (ev) were transfected with pcDNA3.1 empty vector combined with eGFP-containing construct (ratio 10:1).

View larger version (63K): [in this window] [in a new window]   Fig. 4. Both endogenous and exogenous Rac1 and Rac3 localize to different intracellular compartments. (A) N-terminally eGFP-coupled Rac1 or -Rac3 was expressed transiently in N1E-115 cells. Cells were subsequently serum-starved for 8 hours, fixed and stained with phalloidin to visualize filamentous actin. Bar, 25 µm. (B) N1E-115 cells cultured under normal conditions were fixed and stained with anti-Rac1 or anti-Rac3 antibodies. (C) N-terminal eGFP-coupled Rac3*CAAX1 and Rac3*Cterm1 mutants were expressed in N1E-115 cells to monitor their intracellular localization.

View larger version (60K): [in this window] [in a new window]   Fig. 5. Rac3 phenocopies RhoA in N1E-115 cells. (A) Cells were transfected with pcDNA3.1/Rac3, pcDNA3.1/RhoAG14V, pSuper/shRac3 or pSuper/shRhoA, together with 10:1 eGFP. Subsequently, the cells were fixed and stained with phalloidin to visualize F-actin. Bar, 25 µm. Note that both Rac3 and RhoA overexpression induce cell rounding, whereas the depletion of either protein leads to protrusion formation. (B) Morphologies were scored as flat, round or protrusion bearing and shown here in a bar graph. At least 200 cells from two independent experiments were counted. (C) Neurite-like protrusions induced by shRac3 are not responsive to LPA. N1E-115 cells were transfected with either shLuc or shRac3 together with 5:1 eGFP to visualize the transfected cells. Cells were either stimulated or not with 5 µM LPA for 15 minutes and subsequently photographed. (D) Quantification of the results shown in (C). Protrusions-bearing cells were counted in three independent experiments and represented in a bar graph.

View larger version (49K): [in this window] [in a new window]   Fig. 6. Inhibition of ROCK or MLCK impairs RhoA- but not Rac3-induced cell rounding. (A) N1E-115 cells were transfected with control vector (empty vector pcDNA3.1; ev), pcDNA3.1/Rac3 or with pcDNA3/RhoAG14V construct. After being cultured for 48 hours in the presence of 2% serum, cells were treated with the ROCK inhibitor Y-27632 (10 µM) or MLCK inhibitor ML-7 (2 µM) for 4 hours and subsequently photographed. (B) Quantification of the results shown in (A). The round cells were counted (per 100 cells) in at least two different experiments and depicted in a bar graph. (C) In contrast to LPA/RhoA-induced rounding, Rac3-triggered rounding does not involve an increase in MLC phosphorylation (MLC-P). Western blot of N1E-115 cells expressing empty vector, Rac1 or Rac3 that were cultured in 2% serum (lanes 1, 2 and 3). As positive control (Lom et al., 1993), N1E-115 cells were starved during 4 hours and subsequently stimulated with 5 µM LPA for 15 min to induce maximal MLC phosphorylation. As a loading control, both -actin and total MLC levels are shown.

View larger version (71K): [in this window] [in a new window]   Fig. 7. Rac3-expressing cells show defective cell-matrix adhesions, which can be restored by direct activation of integrins by RapG12V. (A) Fluorescent images showing paxillin distribution in N1E-115 cells expressing Rac1 or Rac3. Cells were transiently transfected, cultured in serum-containing medium for 24 hours and subsequently serum-starved for 12 hours to trigger increase in cell-matrix adhesions, which precedes the outgrowth of protrusions and differentiation. Panels on the right show enlargements of the boxed regions in the middle panels. Note the absence of paxillin-rich structures in Rac3-expressing cells (middle and right panel). (B) Depletion of Rac3 is accompanied with increased cell-matrix adhesion and paxillin staining. N1E-115 cells were transfected with shRac3 (together with 5:1 eGFP), cultured for 24 hours and stained for paxillin. (C) Parental N1E-115 cells and N1E-115 cells stably expressing Rac3 were transiently transfected with control vector or pcDNA3.1/RapG12V. Cells were subsequently starved for 1 hour, and stained for paxillin. Note the restoration of the paxillin-rich structures in N1E-115/Rac3 cells that express RapG12V (middle and right panels). (D) Bar graph representing the quantification of the experiment shown in C. At least 100 cells were counted per sample, derived from two independent experiments.

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