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Detection and localization of active GTPases in neuronal cell differentiation
GTPase kits enable powerful signal transduction studies.
Hai Yan Wu, Ph.D.;
Rizwan Farooqui, Ph.D.;
The differentiation of neuronal cells requires signaling pathways that are responsive to the extracellular matrix as well as extensive remodeling of the cytoskeleton. GTPases are critical for these processes. The Ras GTPase family acts as membrane-associated signal transducers; the Rho GTPase family regulates actin and microtubule dynamics. The cellular location of the GTPases and their respective effector binding proteins contribute to cellular differentiation. In this study, Rho and Ras GTPases were assayed for activity and cellular localization using the components of the Thermo Scientific Active GTPase Pull-down and Detection Kits. In addition to using the kits for pull-down assays, the GTPase antibody and GST-tagged effector binding domain were used for immunofluorescent localization. Stimulation of NS-1 cells (neuronal cell line derivative of PC-12) with neuronal growth factor (NGF) resulted in a time- and location-dependent activation of the GTPase targets that were tested. These results could be correlated to staining patterns in primary rat cortical neurons. The kit enabled detection of active GTPase activity in neuronal cells and was an effective tool for studying cellular localization of active GTPase activity.
Differentiated cells have highly specified roles guided by cascades of protein interactions. In these cascades, small GTPases help link cell surface receptors to the actin cytoskeleton, guide interaction with other cells and the extracellular matrix, and direct the delivery and internalization of lipids and proteins. In neurogenesis, cytoskeleton rearrangement and microtubule organization are critical for the initial disruption of cell shape and bud formation for neurite outgrowth and extension. To study neuronal outgrowth in vitro, undifferentiated neuronal cell lines are stimulated with neuronal growth factor (NGF) and monitored for a time period. NGF signaling occurs through the tyrosine kinase receptor (TrkA) and activates Ras GTPase at the membrane. Additional Ras and Rho GTPases are activated after signaling of Ras via PI3 kinase, resulting in active Rap1, RalA and the Rho GTPases. The Ras, Rap1 and RalA GTPases serve as upstream signal transducers; however, the Rho GTPases (Rac1, Cdc42, and RhoA) act antagonistically and affect the actin skeleton and microtubules, transcriptional activation, and membrane trafficking. Rac1 and Cdc42 promote neurite formation and RhoA inhibits neural differentiation. The intricate regulation of GTPases determines the neuronal cell differentiation fate (Figure 1).1-3
Figure 1. Role of GTPases in neurogenesis. A complex balance of interacting signaling pathways controls neuronal cell differentiation. Green arrows: promotes changes in cell morphology; Red lines: abrogates changes in cell morphology.
Regulation of neural differentiation is dependent on both the signaling cascade and the spatial location of the GTPases in context to their respective effector binding proteins. After NGF stimulation, Rac1 is recruited to the membrane to form membrane ruffles and then localizes to the distal half of the neurites during differentiation. Cdc42 is present in the microspikes projecting from the tips. Both Rac1 and Cdc42 trigger neuronal differentiation through Pak1 kinase. If RhoA is activated, RhoA forms a thick ringlike structure at the cell periphery to prevent recruitment of Rac1 to the cell membrane, resulting in neurite retraction.3 The negative regulation of neurite extension by RhoA is dependent on Rho kinase (ROCK). After the initial signaling event that triggers differentiation, each GTPase may induce both positive and negative regulation of neurite growth and axonal signaling (Figure 2) to provide cellular fluidity for signaling and regulatory roles in polarization, extension, guidance and regeneration.
Figure 2. Stages of neuronal development. The GTPases that were assayed in this study are listed below the stages. Green: positive; Red: negative.
We stimulated neuronal NS-1 cells with NGF and studied Rho and Ras family GTPase activity using the Active GTPase Pull-down and Detection Kits. Active GTPase activity was assessed by a functional pull-down assay using a GST fusion of the downstream effector protein that binds only the active form of the GTPase. The spatial distribution of active GTPases was determined by immunofluorescent staining using the GST-PBD protein and anti-GTPase antibody supplied in the kit.
RESULTS and DISCUSSION:
Based on the functional pull-down results, GTPases are differentially regulated with time. Ras activity peaks at day 1 and 2, and Rac1 and Rho activity is present at early time points and diminishes with time. Activity of RalA showed no significant changes (Figure 3). Immunofluorescent staining revealed that Rac1 is present at the membrane ruffles and extends throughout the neurite extension. Colocalization with Pak1 at the periphery and in the neurite tips suggests “active” Rac1 in these regions (Figure 4). Similar staining patterns of Rac1 and Pak1 PBD were obtained with primary differentiated rat cortical neurons (Figure 5). These results loosely correlate with what is reported in earlier studies.1-3 Rho, however, is localized as a thick ring around the cell body and in the perinuclear region. After stimulation, colocalization with rhotekin is in the perinuclear region and does not extend into the neurite extensions, which is consistent with previous results. Ras is also present in the perinuclear region and at the cell periphery, consistent with its function in cell signaling from the membrane to the nucleus. Colocalization with its effector binding domain Raf1 suggests that active Ras is present in the nodes of the neurite extensions as well as in the cell body (Figure 4).
Figure 3. Assay of active GTPase activity by functional pull-down. Active GTPases were detected in NGF-stimulated NS-1 cells as described in Methods. Spot densitometry was performed on each scanned blot and normalized to scale. The graph summarizes the induction of Ras, Rac1, Rho, and RalA for a four day period.
Figure 4. Use of GTPase binding domains as antibody alternatives localizes GTPases activity in differentiated neuronal cells. NS-1 cells were grown and treated with NGF as described in Methods. Images of GTPase and GTPasebinding domain (BD) staining of day two differentiated cells are monochrome. Merged images of non-treated (control) and day two differentiated cells are multicolored. GTPases were detected using DyLight 549-conjugated secondary antibodies. The GTPase effector binding proteins were detected using DyLight 488-conjugated anti-GST antibody. Arrows denote areas of colocalization.
Figure 5. Rac1 and Pak1 exhibit similar localization patterns in rat primary cortical neurons. Fresh cortex tissue from E18 Sprague-Dawley rat (BrainBits, Springfield, IL) was used to culture primary neurons in poly-D-lysine-coated 12-well slides. Cells were co-stained with Rac1 and Pak1 as described in Methods.
Using the Active GTPase Pull-down and Detection Kits enabled visualization of GTPase activity during the course of differentiation and GTPase cellular localization. The GST-tagged GTPase effector binding domain should only stain “active GTPases”; however, some of the effectors bind multiple proteins and GTPases. Therefore, co-localization of the GTPase antibody with the respective binding domain suggests the cellular location of “active” GTPases. Co-localization studies provide a better understanding of spatial activity during differentiation; however, more optimization of the effector binding domains is necessary for better specificity and lower background.
NS-1 cells were cultured in RPMI media containing 15% FBS, pen/strep, and HEPES buffer on collagen IV-coated plates or on cell culture-treated eight-well chamber slides (BD Biosciences). At ~80% confluency, cells were stimulated with 50ng/mL NGF (EMD Biosciences) or untreated. For functional pull-down assays, cells were harvested at 1 hour and 1, 2, 3, and 4 days post-treatment using the lysis buffer supplied in the kit. Active GTPases were detected from fresh cell lysates (1mg total protein) by Western blot as per kit instructions. For immunofluorescent staining, media was gently removed and replaced with warmed 4% paraformaldehyde for 20-30 minutes at 37˚C, to preserve neuronal structure. Slides were stored at 4˚C until stained.
After fixation, cells were permeabilized with 0.05% Triton* X-100 in phosphate-buffered saline (PBS) for 15 minutes, and blocked for 30 minutes in 5% fetal bovine serum (FBS) in PBS. Cells were incubated with the GST-effector binding domain fusion protein (Raf1, rhotekin, or Pak1) at 50-200μg/mL for 1 hour, rinsed and stained with anti-GTPase antibody (Ras, pan-Rho and Rac1, respectively) for 1 hour at room temperature (1:250-1:500 dilution). Cells were washed and stained with Thermo Scientific DyLight 549 Dye conjugated to goat anti-mouse or goat anti-rabbit IgG (1:500 dilution), DyLight 488 Dye conjugated to anti-GST antibody (1:500 dilution) and Hoechst 33342 (DNA stain) for 30 minutes at room temperature. Cells were washed and dehydrated using a 70, 80, 90, 100 ethanol series. Coverslips were mounted using VECTASHIELD* Mounting Media, and images were acquired using the Axio Observer (Carl-Zeiss, Inc.) inverted microscope (63X objective) and AxioVision Software Module.
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