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In Vitro Assay of Primary Astrocyte Migration as a Tool to Study Rho GTPase Function in Cell Polarization
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Khosravi‐Far, R., Solski, P. A., Clark, G. J., Kinch, M. S., and Der, C. J. (1995). Activation of Rac1, RhoA, and mitogen‐activated protein kinases is required for Ras transformation. Mol. Cell. Biol. 15, 6443–6453. Rarey, M., Kramer, B., Lengauer, T., and Klebe, G. (1996). A fast flexible docking method using an incremental construction algorithm. J. Mol. Biol. 261, 470–489. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992). The small GTP‐binding protein rac regulates growth factor‐induced membrane ruffling. Cell 70, 401–410. Schmidt, A., and Hall, A. (2002). Guanine nucleotide exchange factors for Rho GTPases: Turning on the switch. Gene Dev. 16, 1587–1609. Soulet, C., Gendreau, S., Missy, K., Benard, V., Plantavid, M., and Payrastre, B. (2001). Characterisation of Rac activation in thrombin‐ and collagen‐stimulated human blood platelets. FEBS Lett. 507, 253–258. Vidal, C., Geny, B., Melle, J., Jandrot‐Perrus, M., and Fontenay‐Roupie, M. (2002). Cdc42/ Rac1‐dependent activation of the p21‐activated kinase (PAK) regulates human platelet lamellipodia spreading: Implication of the cortical‐actin binding protein cortactin. Blood 100, 4462–4469. Worthylake, D. K., Rossman, K. L., and Sondek, J. (2000). Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1. Nature 408, 682–688. Yang, F. C., Atkinson, S. J., Gu, Y., Borneo, J. B., Roberts, A. W., Zheng, Y., Pennington, J., and Williams, D. A. (2001). Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization. Proc. Natl. Acad. Sci. USA 98, 5614–5618. Zheng, Y. (2001). Dbl family guanine nucleotide exchange factors. Trends Biochem. Sci. 26, 724–732.
[44] In Vitro Assay of Primary Astrocyte Migration as a Tool to Study Rho GTPase Function in Cell Polarization By SANDRINE ETIENNE‐MANNEVILLE Abstract
Rho GTPases are key players in cell migration. The contribution of Rho, Rac, and Cdc42 to the regulation of the actin and microtubule cytoskeletons is essential for membrane protrusion and cell retraction (Etienne‐Manneville and Hall, 2002). The polarization of these protrusive and retracting activities in a migrating cell is also under the control of Rho GTPases, in particular Cdc42 (Nobes and Hall, 1999). In vitro study of cell migration has shown that Cdc42 activity is required for polarized cell migration in several cell types, including fibroblasts, neutrophils, macrophages, and astrocytes (Allen et al., 1998; Etienne‐Manneville, 2004; Etienne‐Manneville and Hall, 2001; Palazzo et al., 2001; Srinivasan et al., 2003). Using scratch‐induced migration assay, we have previously used primary astrocytes as a tool to study the molecular mechanisms controlling METHODS IN ENZYMOLOGY, VOL. 406 Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(06)06044-7
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cell polarization at the onset of migration (Etienne‐Manneville and Hall, 2001, 2003). On scratching of the monolayer, astrocytes polarize perpendicularly to the scratch to migrate and close the wound. Astrocyte polarization is characterized by the formation of a protrusion in the direction of migration, the elongation of the microtubules that fill the protrusion, and the reorientation of the centrosome, which serves as a microtubule‐organizing center toward the direction of migration. This in vitro migration assay allows us to simultaneously investigate the mechanisms controlling cell migration, cell protrusion, and cell polarization. Primary astrocytes, although more constraining, provide a more physiological model than immortalized cell lines. Moreover, astrocyte culture can be obtained in a large number and, therefore, also allows biochemical analysis. Here I describe the procedure by which we can obtain and purify primary rat astrocytes and the different assays we have previously used to analyze the role of Rho GTPases and their downstream targets in cell migration and polarization. Introduction
Astrocytes are the most abundant cells in the central nervous system (CNS) with intricate relationships with neurons, blood vessels, and meninges. The ratio of astrocytes to neurons has increased during evolution. Although astrocytes have been traditionally viewed as neuron‐supporting cells, evidence is now accumulating that astrocytes play a wide range of functions critical for maintenance of a homeostatic neuronal environment and are also directly involved in neuronal development and functions (Haydon, 2000; Song et al., 2002; Ullian et al., 2001). Astrocytes are suspected to be involved in a wide range of CNS pathologies, including trauma, viral, or bacterial infections but also neurodegeneration (Mucke and Eddleston, 1993). In such situations, astrocytes undergo a reaction called astrogliosis with increased cell division and protein expression and a characteristic morphological change (the hypertrophy of their cellular processes) associated with an increased motility (Ridet et al., 1997). The poor ability of mammalian central nervous system axons to regenerate has been attributed, in part, to astrocyte behavior after axonal injury (Shearer and Fawcett, 2001; Sivron and Schwartz, 1995). This behavior is manifested by the limited ability of astrocytes to migrate and thus repopulate the site of injury. The slow repopulation of the injury site by astrocytes is in apparent correlation with the failure of the regrowing injured axons to traverse this site (Rhodes et al., 2003; Silver and Miller, 2004). Understanding why mammalian astrocytes migrate or fail to repopulate the site of
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injury and finding ways to overcome this failure may contribute significantly to the achievement of CNS regeneration. Although connected by gap junctions, astrocytes do not form any defined structure amenable to experimental manipulation, and, therefore, astrocytes have been relatively difficult to study in vivo. The development of in vitro models of astrocyte migration has provided a useful tool to investigate the molecular mechanisms controlling astrocyte migration. Our experimental procedure was derived from ones used to follow gliosis in response to in vitro injury or the effects of wound‐associated factors on astrocyte migratory response (Faber‐Elman et al., 1996) and recapitulate most of the major astrocyte responses to in vivo injury. In addition to their physiological relevance, primary astrocytes have appeared as a beautiful cell biology model. Using this scratch‐induced in vitro migration assay, we have observed that astrocytes undergo slow and polarized migration in the direction perpendicular to the scratch (Etienne‐Manneville and Hall, 2001). Cells do not move individually but rather migrate as a sheet toward the other edge of the scratch. Migration stops when the two edges of the scratch meet. This assay has proved to be a very powerful tool to investigate the molecular mechanisms controlling cell migration and cell polarization (Etienne‐Manneville and Hall, 2001, 2003). Confluent astrocytes are barely motile, and migration is initiated on scratching. Migrating astrocytes are morphologically different from immobile cells. They are extremely flat, firmly adherent, and with an elongated protrusion easily reaching 100 m long. Indeed, on scratching, the leading edge protrudes continuously for at least 12 h, forming an elongated protrusion extending in the direction of migration and reminiscent of the large astrocytic process visible in vivo. The protrusion is filled with elongated microtubules. Protrusion formation, as well as migration, is dependent on microtubule dynamics, which makes it a valuable model to study the regulation of the microtubule network. As part of microtubule network rearrangement, the centrosome reorients in front of the nucleus in the direction of cell migration. Although not always required for migration, centrosome reorientation is a common theme in many migrating cells, including fibroblasts, epithelial cells, and neurons (Magdalena et al., 2003; Palazzo et al., 2001; Yvon et al., 2002). In astrocytes, centrosome position seems to be a good indicator for the orientation of the protrusion and, therefore, reflects the orientation of the polarized cells. Therefore, protrusion formation and centrosome reorientation are two essential markers of polarization of migrating astrocytes, which can be used to investigate the pathways involved in cell polarization.
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Primary Culture of Rat Cerebral Astrocytes
Reagents Dissection buffer (DB): PBS (without calcium and magnesium), 0.6% glucose. Astrocyte culture medium (ACM): DMEM, 1 g/l glucose, 2 mM L‐glutamine, 100 mg/ml penicillin/streptomycin, 10% heat‐inactivated fetal calf serum. Poly‐L‐ornithine hydrobromide (Sigma‐Aldrich). Dissection Procedure To purify primary astrocytes we use a method derived from El‐Etr et al. (1989) and Weiss et al. (1986) 1. An 18‐day pregnant Sprague‐Dawley female rat is put to death in a CO2 chamber according to published recommendations (Close et al., 1996a,b). 2. Embryos are collected in a Petri dish containing cold DB and placed on ice. 3. Embryos are decapitated and heads moved to a second Petri dish containing cold DB and placed on ice. 4. While steadying the embryo head with forceps, cut the skin and the skull from the midline of the head at the base of the skull to the eyes with microdissecting curved scissors. 5. If necessary, cut the skull at the midline fissure, without cutting into the brain tissue, to facilitate brain extraction. 6. Release the brain from the skull cavity. Apply slight pressure with microdissecting curved forceps, sliding along the length of the brain from the mid‐eye area backward to the base of the skull. 7. Using small curved forceps, transfer each brain to a 60‐mm Petri dish containing cold DB and placed on ice. Leave on ice approximatively 15 min. Use this time to coat tissue culture plates with polyornithine (see ‘‘Tissue Culture Plate Coating’’). 8. While steadying the brain with forceps, separate the cerebrum from the cerebellum and the brain stem. 9. Cut with fine microdissecting scissors on each sides of the midline fissure. 10. Gently peel the meninges from individual cortical lobes. 11. Dissect out each striatum and immediately transfer it to a 35‐mm Petri dish containing 2 ml of cold serum‐free ACM.
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12. Once all the striata have been transferred to the 35‐mm Petri dish, check the absence of remaining meninges. 13. Cut striata in pieces and transfer them with the 2 ml of serum‐free ACM to a 15‐ml tube. 14. Mechanically dissociate into a cell suspension with a narrow Pasteur pipette by pipetting up and down at least 15 times. 15. Add 2 ml of serum‐free ACM and mix by gentle inversion. 16. Decant for 5 min. 17. Transfer the top 2 ml in a 50‐ml tube containing 37 preheated ACM 37 and keep it in the 37 incubator. 18. Add 1 ml of serum‐free ACM. 19. Repeat steps 4–6 using a narrower Pasteur pipette. 20. After decantation, pool the cell suspension with the previous one and discard the remaining cell agglomerates at the bottom of the tube. You should not have to discard more than 100–200 l. If cells have not been sufficiently dissociated, go back to step 19. 21. Centrifuge at low speed (800 rpm) for 8 min. 22. Discard the supernatant and resuspend the pellet in ACM. 23. Count the cells and dilute them to 3 105 cells/ml in the appropriate final volume of preheated ACM. 24. Keep the cells at 37 while washing the tissue culture plates (see ‘‘Tissue Culture Plate Coating’’). 25. Plate 10 ml (3 106 cells) of cells in poly‐L‐ornithine–coated 90‐mm tissue culture dishes. 26. Place the tissue culture dishes at 37 in a moist 5% CO2, 95% air atmosphere. Tissue Culture Plate Coating You should start the coating before having finished the dissection procedure, so that you do not have to keep the cells in suspension in the incubator before plating. 1. As a first estimation, you can coat one 90‐mm tissue culture Petri dish per embryo. Add another three to four plates in case you get more cells at the end of the dissection. 2. Prepare a 1.5 g/ml poly‐L‐ornithine solution in sterile distilled water. 3. Use 4 ml of poly‐L‐ornithine solution for each 90‐mm tissue culture Petri dish. 4. Incubate the plates for 1 h at 37 . 5. Discard the poly‐L‐ornithine solution.
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6. Wash the plates once with 10 ml of water. Culture Purification and Differentiation 1. Keep the cells in the incubator for 7 days without changing the medium. 2. After 7 days, remove the medium and replace it with 10 ml PBS. 3. Seven days after dissection, only few astrocytes are visible; most cells are neural precursors. 4. Wash thoroughly the entire cell monolayer by flushing 10 times PBS directly onto the cells. This should separate remaining oligodendrocytes and microglial cells; remove all debris, dead cells, and aggregates from the plates. 5. Replace the PBS with 10 ml of fresh ACM. Return the plates to the 37 incubator for 48 h. 6. Change the medium every 2 days until cells form a confluent monolayer. Cells usually form a confluent monolayer 10–12 days after dissection. Confluent plates contain 4 106 to 5 106 cells. 7. After 3 weeks in culture, routinely more than 95% of the cells are positively stained for glial fibrillary acidic protein (GFAP). 8. Confluent cells can then be kept for 2 weeks (changing medium twice a week) or be passed once onto poly‐L‐ornithine–coated plates or glass coverslip for further enrichment. 9. For better results, astrocytes must be passaged during the first week after confluency. 10. Wash cells in calcium‐ and magnesium‐free PBS. 11. Add 1 ml of trypsin 0.25%, EDTA 0.02% per 90‐mm tissue culture dish and incubate for 5 min at 37 . 12. Detach the cells by flushing the trypsin solution on the monolayer, add the cell suspension in a 15‐ml tube containing 14 ml of ACM and centrifuge for 8 min at 800 rpm. 13. Resuspend the cell pellet in the appropriate ACM volume, so that cell density/surface is divided three times. (One 90‐mm dish will give rise to three 90‐mm dishes of confluent astrocytes or to the equivalent surface on glass coverslips). 14. Secondary cultures are grown to confluency changing medium every 48 h and can be kept for a further 2–3 weeks changing medium twice a week. 15. We do not recommend a second passage.
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Scratch‐Induced Astrocyte Migration
Astrocytes like many other cell types will undergo migration after scratching of the cell monolayer (Fig. 1). Therefore, cell migration will occur whether you use fully differentiated astrocytes or not. However, astrocyte‐specific responses, in particular astrocyte protrusion, will occur only if the cells are fully differentiated. In vitro differentiation of astrocytes can be monitored by the nature of the intermediate filaments. Differentiated astrocytes are characterized by GFAP expression (Eliasson et al., 1999; Pekny and Pekna, 2004). Regular GFAP staining is recommended. Because the number of differentiated astrocytes increases while the cells are maintained in culture as confluent monolayers, the longer you wait the more differentiated they will be. So, we recommend waiting at least 7 days after confluence before performing a scratch assay.
FIG. 1. Scratch‐induced migration of primary rat astrocytes. Phase‐contrast images from a video showing scratch‐induced astrocyte migration. The time period after scratching is indicated at the bottom of each picture. Note the morphological changes (protrusion formation) occurring during the first 12 h and the nuclear movement only seen later. Bar, 100 m.
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Single Wound Procedure for Cell Biology Analysis 1. Use a confluent monolayer of astrocytes plated on a glass coverslip (diameter, 12–14 mm) placed in a 4‐well plate. 2. Change the cell culture medium 16 or 24 h before scratching. 3. Make a single scratch in the monolayer by moving a flamed glass capillary at regular speed across the glass coverslip. Depending on the size of the glass capillaries, the width of the scratch will vary. Because astrocytes migrate relatively slowly, scratches 300‐mm wide are sufficient for most experiments. However, for migration assays, use large glass capillaries or Pasteur pipette to make wide scratch (500 m). 4. Do not change the medium and return the coverslip in the 4‐well plate in the incubator for the desired time period. Multiple Wound Procedure for Biochemical Analysis To assess regulation of enzymatic activity (kinase assays), changes in protein content, GTP‐loading of GTPases, or protein complex formation, you must first obtained a large number of migrating astrocytes. To scale up the number of scratch‐activated cells, a large number of scratches is done on large plate of confluent cells. 1. Use confluent monolayers of astrocytes plated on 35‐mm dishes or 90‐mm dishes. 2. Change the cell culture medium 16 h before scratching. 3. Make multiple scratches in the monolayer so that at least 50% of the cells are on the edge of a scratch. We recommend the use of an eight‐channel pipet with 1–10 l tips (three tips are enough to scratch a 35‐mm dish). Slide the eight tips altogether three times around the plate and 10 times across it turning the plate a 20‐degree angle each time. 4. Incubate the plate for the required amount of time in a 37 , 5% CO2 incubator. Analysis of Scratch‐Induced Astrocyte Responses
Procedure for Immunostaining 1. Wash cells twice with 1 ml PBS. 2. Fix cells with 250 l PBS, paraformaldehyde 4%, (supplemented with glutaraldehyde 0.5% for microtubules or actin filament staining) for 10 min at room temperature.
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3. Wash twice with 1 ml PBS. 4. Permeabilize the cells with PBS, Triton 0.2%, for 10 min at room temperature. 5. Wash twice with 1 ml PBS. 6. Quench for 10 min at room temperature with 2 mg/ml NaBH4 in PBS. 7. Wash twice with 1 ml PBS. 8. Incubate with primary antibodies for 45 min at room temperature. 9. Wash 10 times in PBS. 10. Incubate with labeled‐secondary antibodies for 30 min at room temperature. 11. Wash 10 times with PBS and twice more with H2O. 12. Mount the coverslip in 5 l of Mowiol (Calbiochem) or other mounting reagent. Let the Mowiol dry for 30 min at 37 or overnight at 4 .
Astrocyte Migration Assay One must be careful not to assess cell protrusion instead of cell translocation. Indeed, the cell leading edge movement is not representative of the movement of the cell rear. Nuclear migration is barely visible during the first 6 h, and translocation of the cell body starts 8–12 h after scratching, while protrusions have already invaded the space left by the scratch (Fig. 2A). Therefore, we strongly recommend assessment of nuclear migration as a valuable sign of astrocyte migration rather than the closure of the wound, which reflects both migration and protrusion. 1. Make a large wound (approximately 500‐mm wide) across the astrocyte monolayer plated on a 12‐ to 14‐mm coverslip. 2. Immediately microinject pEGFP vector in the nucleus of cells on the edge of the scratch. 3. Place the cells back in the 37 , 5% CO2 incubator and leave them to migrate for 48 h. 4. Wash cells twice with 1 ml PBS. 5. Incubate the cells with Hoechst solution for 10 min at room temperature. Hoechst staining is sufficient to assess astrocyte migration; however, staining for actin, microtubules, or any protein of interest can be performed on the same cells. In this case, use the immunostaining procedure accordingly. 6. Wash four times in PBS. 7. Mount the cells by inverting the glass coverslip on a microscope slide on which you will have put 5 l Mowiol.
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8. Observe under the microscope at low magnification (10 or 20 objectives). Looking at the Hoechst staining, you will easily recognize the initial position of the scratch as a line with more numerous (corresponding to the accumulation of nonmigrating cells) and condensed nuclei (which mark dead cells left after scratching) (Fig. 2A, right panel). Find the GFP‐expressing cells and among these score as migrating cells those cells whose nuclei have moved away from their original place. Do not expect all expressing cells to have migrated. Approximatively only 60% of expressing cells are migrating cells. An alternative (and more fastidious) way to test astrocyte migration is to do a time‐lapse movie for 36–48 h after scratching and to assess nuclear migration by tracking the nuclei of wound‐edge cells during wound closure. Quantification of Astrocyte Protrusion We have defined astrocyte protrusion as a morphological characteristic of migrating astrocytes (Etienne‐Manneville and Hall, 2001). Protrusion starts during the first hour after scratching (Fig. 2B). However, for the first 4–6 h, the protrusion is comparable in shape and length to a fibroblast lamellipodia. It is only after 8 h that the elongation of the protrusion becomes evident. The protrusion being very flat, we recommend the use of microtubule staining (Fig. 2B). Indeed, microtubules elongate in the entire protrusion and allow easy measurement of the cell length. Protrusion formation can, therefore, be assessed 8 h after wounding; however, for easier quantification, we generally score the number of protrusions formed 16 h after scratching. Except for the migration time, the protocol is identical in both cases. 1. Make a wound (approximately 300‐mm wide) across the astrocyte monolayer plated on a 12‐ to 14‐mm coverslip.
FIG. 2. Scratch‐induced astrocyte responses. (A) Cell migration. Left panel shows tubulin (microtubules, green) and Hoechst (nuclei, blue) 24 h after scratching. From the Hoechst image shown on the right panel, the initial position of the scratch can be drawn (red line). Bar, 100 m. (B) Protrusion formation and polarization of the microtubule network. Staining with antitubulin antibodies at indicated time after scratching shows the elongation of the microtubule network and its polarization along the axis of migration. Bar, 10 m. (C) Centrosome reorientation. Concomitant Hoechst (nuclei, blue) and pericentrin (centrosome, red) staining allow the quantification of centrosome reorientation. In the left panel (0 h after scratching), the two cells of the front row are not polarized, their centrosome is not localized in the forward‐facing quadrant. In the right panel (8 h after scratching), the centrosomes of the scratch edge cells are localized in the forward‐facing quadrant. Bar, 10 m.
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2. Immediately microinject pEGFP vector or your vector of interest in the nucleus of cells of the wound edge. 3. Place the cells back in the 37 , 5% CO2 incubator and leave them to migrate for 16 h. 4. Wash cells twice with 1 ml PBS. 5. Use the immunostaining procedure with antibodies directed against tubulin. 6. Observe under the fluorescence microscope using (20 or 40 objective). Find the GFP/protein of interest expressing cells. Among these cells, looking at the microtubule staining, score as ‘‘protruding’’ cells with one major protrusion that appear at least four times longer than wide. Do not expect all expressing cells to have protrusions. Approximately 65% of GFP‐expressing cells form protrusions. We score protruding cells regardless of the orientation of the protrusion. When polarization is inhibited in the entire monolayer (for instance with PKC or GSK3 inhibitors (Etienne‐ Manneville and Hall, 2001, 2003), the number of protruding cells remains normal; however, protrusions form in random directions, and smaller protrusions can be seen on the sides of the major protrusion. Centrosome Reorientation Assay The centrosome reorientation assay allows a precise quantification of cell orientation (Fig. 2C). In astrocytes, the centrosome also serves as the major microtubule organizing center, where most microtubule minus‐ends are anchored. In a confluent astrocyte monolayer, the centrosome is localized in close proximity to the nuclear envelope in a random position around the nucleus. On scratching, the centrosome of most cells (about 75%, 16 h after scratching) localizes in front of the nucleus toward the front edge (Etienne‐Manneville and Hall, 2001). Centrosome reorientation essentially occurs during the first 6–8 h after scratching and before cell translocation. 1. For centrosome reorientation assay, allow the cells to migrate for 8 h. 2. Wash cells twice with 1 ml PBS. 3. Use the immunostaining procedure with antibodies directed against pericentrin and Hoechst. 4. Observe under the microscope using 63 objective. Centrosome reorientation must be assessed in the cells located at the wound edge
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(Fig. 2C). Eight hours after wounding, the centrosome of cells in the second and third rows of cells is not reoriented yet. To quantify centrosome reorientation, separate the space in four equal quadrants joining in the center of the nucleus of interest and placed so that one of the quadrant is facing the scratch (the median of each 90‐ degree angle being either parallel or perpendicular to the scratch) (Fig. 2C). Do not try to determine centrosome orientation along curved or irregular portions of the scratch. Centrosomes are scored as ‘‘polarized’’ or ‘‘reoriented’’ when the centrosome is placed in the quadrant facing the scratch. When cells have two centrosomes (i.e., cells in G2), centrosomes are scored polarized if one of the two centrosomes is correctly positioned. Note that in a nonpolarized population of cells, such as wound edge cells just after wounding, 25% of the centrosome will be positively scored; 25% of ‘‘polarized’’ centrosome, therefore, corresponds to a random cell orientation. Eight hours after wounding, you can expect approximately 65% of ‘‘polarized’’ centrosomes.
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Khosravi‐Far, R., Solski, P. A., Clark, G. J., Kinch, M. S., and Der, C. J. (1995). Activation of Rac1, RhoA, and mitogen‐activated protein kinases is required for Ras transformation. Mol. Cell. Biol. 15, 6443–6453. Rarey, M., Kramer, B., Lengauer, T., and Klebe, G. (1996). A fast flexible docking method using an incremental construction algorithm. J. Mol. Biol. 261, 470–489. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992). The small GTP‐binding protein rac regulates growth factor‐induced membrane ruffling. Cell 70, 401–410. Schmidt, A., and Hall, A. (2002). Guanine nucleotide exchange factors for Rho GTPases: Turning on the switch. Gene Dev. 16, 1587–1609. Soulet, C., Gendreau, S., Missy, K., Benard, V., Plantavid, M., and Payrastre, B. (2001). Characterisation of Rac activation in thrombin‐ and collagen‐stimulated human blood platelets. FEBS Lett. 507, 253–258. Vidal, C., Geny, B., Melle, J., Jandrot‐Perrus, M., and Fontenay‐Roupie, M. (2002). Cdc42/ Rac1‐dependent activation of the p21‐activated kinase (PAK) regulates human platelet lamellipodia spreading: Implication of the cortical‐actin binding protein cortactin. Blood 100, 4462–4469. Worthylake, D. K., Rossman, K. L., and Sondek, J. (2000). Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1. Nature 408, 682–688. Yang, F. C., Atkinson, S. J., Gu, Y., Borneo, J. B., Roberts, A. W., Zheng, Y., Pennington, J., and Williams, D. A. (2001). Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization. Proc. Natl. Acad. Sci. USA 98, 5614–5618. Zheng, Y. (2001). Dbl family guanine nucleotide exchange factors. Trends Biochem. Sci. 26, 724–732.
[44] In Vitro Assay of Primary Astrocyte Migration as a Tool to Study Rho GTPase Function in Cell Polarization By SANDRINE ETIENNE‐MANNEVILLE Abstract
Rho GTPases are key players in cell migration. The contribution of Rho, Rac, and Cdc42 to the regulation of the actin and microtubule cytoskeletons is essential for membrane protrusion and cell retraction (Etienne‐Manneville and Hall, 2002). The polarization of these protrusive and retracting activities in a migrating cell is also under the control of Rho GTPases, in particular Cdc42 (Nobes and Hall, 1999). In vitro study of cell migration has shown that Cdc42 activity is required for polarized cell migration in several cell types, including fibroblasts, neutrophils, macrophages, and astrocytes (Allen et al., 1998; Etienne‐Manneville, 2004; Etienne‐Manneville and Hall, 2001; Palazzo et al., 2001; Srinivasan et al., 2003). Using scratch‐induced migration assay, we have previously used primary astrocytes as a tool to study the molecular mechanisms controlling METHODS IN ENZYMOLOGY, VOL. 406 Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(06)06044-7
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cell polarization at the onset of migration (Etienne‐Manneville and Hall, 2001, 2003). On scratching of the monolayer, astrocytes polarize perpendicularly to the scratch to migrate and close the wound. Astrocyte polarization is characterized by the formation of a protrusion in the direction of migration, the elongation of the microtubules that fill the protrusion, and the reorientation of the centrosome, which serves as a microtubule‐organizing center toward the direction of migration. This in vitro migration assay allows us to simultaneously investigate the mechanisms controlling cell migration, cell protrusion, and cell polarization. Primary astrocytes, although more constraining, provide a more physiological model than immortalized cell lines. Moreover, astrocyte culture can be obtained in a large number and, therefore, also allows biochemical analysis. Here I describe the procedure by which we can obtain and purify primary rat astrocytes and the different assays we have previously used to analyze the role of Rho GTPases and their downstream targets in cell migration and polarization. Introduction
Astrocytes are the most abundant cells in the central nervous system (CNS) with intricate relationships with neurons, blood vessels, and meninges. The ratio of astrocytes to neurons has increased during evolution. Although astrocytes have been traditionally viewed as neuron‐supporting cells, evidence is now accumulating that astrocytes play a wide range of functions critical for maintenance of a homeostatic neuronal environment and are also directly involved in neuronal development and functions (Haydon, 2000; Song et al., 2002; Ullian et al., 2001). Astrocytes are suspected to be involved in a wide range of CNS pathologies, including trauma, viral, or bacterial infections but also neurodegeneration (Mucke and Eddleston, 1993). In such situations, astrocytes undergo a reaction called astrogliosis with increased cell division and protein expression and a characteristic morphological change (the hypertrophy of their cellular processes) associated with an increased motility (Ridet et al., 1997). The poor ability of mammalian central nervous system axons to regenerate has been attributed, in part, to astrocyte behavior after axonal injury (Shearer and Fawcett, 2001; Sivron and Schwartz, 1995). This behavior is manifested by the limited ability of astrocytes to migrate and thus repopulate the site of injury. The slow repopulation of the injury site by astrocytes is in apparent correlation with the failure of the regrowing injured axons to traverse this site (Rhodes et al., 2003; Silver and Miller, 2004). Understanding why mammalian astrocytes migrate or fail to repopulate the site of
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injury and finding ways to overcome this failure may contribute significantly to the achievement of CNS regeneration. Although connected by gap junctions, astrocytes do not form any defined structure amenable to experimental manipulation, and, therefore, astrocytes have been relatively difficult to study in vivo. The development of in vitro models of astrocyte migration has provided a useful tool to investigate the molecular mechanisms controlling astrocyte migration. Our experimental procedure was derived from ones used to follow gliosis in response to in vitro injury or the effects of wound‐associated factors on astrocyte migratory response (Faber‐Elman et al., 1996) and recapitulate most of the major astrocyte responses to in vivo injury. In addition to their physiological relevance, primary astrocytes have appeared as a beautiful cell biology model. Using this scratch‐induced in vitro migration assay, we have observed that astrocytes undergo slow and polarized migration in the direction perpendicular to the scratch (Etienne‐Manneville and Hall, 2001). Cells do not move individually but rather migrate as a sheet toward the other edge of the scratch. Migration stops when the two edges of the scratch meet. This assay has proved to be a very powerful tool to investigate the molecular mechanisms controlling cell migration and cell polarization (Etienne‐Manneville and Hall, 2001, 2003). Confluent astrocytes are barely motile, and migration is initiated on scratching. Migrating astrocytes are morphologically different from immobile cells. They are extremely flat, firmly adherent, and with an elongated protrusion easily reaching 100 m long. Indeed, on scratching, the leading edge protrudes continuously for at least 12 h, forming an elongated protrusion extending in the direction of migration and reminiscent of the large astrocytic process visible in vivo. The protrusion is filled with elongated microtubules. Protrusion formation, as well as migration, is dependent on microtubule dynamics, which makes it a valuable model to study the regulation of the microtubule network. As part of microtubule network rearrangement, the centrosome reorients in front of the nucleus in the direction of cell migration. Although not always required for migration, centrosome reorientation is a common theme in many migrating cells, including fibroblasts, epithelial cells, and neurons (Magdalena et al., 2003; Palazzo et al., 2001; Yvon et al., 2002). In astrocytes, centrosome position seems to be a good indicator for the orientation of the protrusion and, therefore, reflects the orientation of the polarized cells. Therefore, protrusion formation and centrosome reorientation are two essential markers of polarization of migrating astrocytes, which can be used to investigate the pathways involved in cell polarization.
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Primary Culture of Rat Cerebral Astrocytes
Reagents Dissection buffer (DB): PBS (without calcium and magnesium), 0.6% glucose. Astrocyte culture medium (ACM): DMEM, 1 g/l glucose, 2 mM L‐glutamine, 100 mg/ml penicillin/streptomycin, 10% heat‐inactivated fetal calf serum. Poly‐L‐ornithine hydrobromide (Sigma‐Aldrich). Dissection Procedure To purify primary astrocytes we use a method derived from El‐Etr et al. (1989) and Weiss et al. (1986) 1. An 18‐day pregnant Sprague‐Dawley female rat is put to death in a CO2 chamber according to published recommendations (Close et al., 1996a,b). 2. Embryos are collected in a Petri dish containing cold DB and placed on ice. 3. Embryos are decapitated and heads moved to a second Petri dish containing cold DB and placed on ice. 4. While steadying the embryo head with forceps, cut the skin and the skull from the midline of the head at the base of the skull to the eyes with microdissecting curved scissors. 5. If necessary, cut the skull at the midline fissure, without cutting into the brain tissue, to facilitate brain extraction. 6. Release the brain from the skull cavity. Apply slight pressure with microdissecting curved forceps, sliding along the length of the brain from the mid‐eye area backward to the base of the skull. 7. Using small curved forceps, transfer each brain to a 60‐mm Petri dish containing cold DB and placed on ice. Leave on ice approximatively 15 min. Use this time to coat tissue culture plates with polyornithine (see ‘‘Tissue Culture Plate Coating’’). 8. While steadying the brain with forceps, separate the cerebrum from the cerebellum and the brain stem. 9. Cut with fine microdissecting scissors on each sides of the midline fissure. 10. Gently peel the meninges from individual cortical lobes. 11. Dissect out each striatum and immediately transfer it to a 35‐mm Petri dish containing 2 ml of cold serum‐free ACM.
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12. Once all the striata have been transferred to the 35‐mm Petri dish, check the absence of remaining meninges. 13. Cut striata in pieces and transfer them with the 2 ml of serum‐free ACM to a 15‐ml tube. 14. Mechanically dissociate into a cell suspension with a narrow Pasteur pipette by pipetting up and down at least 15 times. 15. Add 2 ml of serum‐free ACM and mix by gentle inversion. 16. Decant for 5 min. 17. Transfer the top 2 ml in a 50‐ml tube containing 37 preheated ACM 37 and keep it in the 37 incubator. 18. Add 1 ml of serum‐free ACM. 19. Repeat steps 4–6 using a narrower Pasteur pipette. 20. After decantation, pool the cell suspension with the previous one and discard the remaining cell agglomerates at the bottom of the tube. You should not have to discard more than 100–200 l. If cells have not been sufficiently dissociated, go back to step 19. 21. Centrifuge at low speed (800 rpm) for 8 min. 22. Discard the supernatant and resuspend the pellet in ACM. 23. Count the cells and dilute them to 3 105 cells/ml in the appropriate final volume of preheated ACM. 24. Keep the cells at 37 while washing the tissue culture plates (see ‘‘Tissue Culture Plate Coating’’). 25. Plate 10 ml (3 106 cells) of cells in poly‐L‐ornithine–coated 90‐mm tissue culture dishes. 26. Place the tissue culture dishes at 37 in a moist 5% CO2, 95% air atmosphere. Tissue Culture Plate Coating You should start the coating before having finished the dissection procedure, so that you do not have to keep the cells in suspension in the incubator before plating. 1. As a first estimation, you can coat one 90‐mm tissue culture Petri dish per embryo. Add another three to four plates in case you get more cells at the end of the dissection. 2. Prepare a 1.5 g/ml poly‐L‐ornithine solution in sterile distilled water. 3. Use 4 ml of poly‐L‐ornithine solution for each 90‐mm tissue culture Petri dish. 4. Incubate the plates for 1 h at 37 . 5. Discard the poly‐L‐ornithine solution.
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6. Wash the plates once with 10 ml of water. Culture Purification and Differentiation 1. Keep the cells in the incubator for 7 days without changing the medium. 2. After 7 days, remove the medium and replace it with 10 ml PBS. 3. Seven days after dissection, only few astrocytes are visible; most cells are neural precursors. 4. Wash thoroughly the entire cell monolayer by flushing 10 times PBS directly onto the cells. This should separate remaining oligodendrocytes and microglial cells; remove all debris, dead cells, and aggregates from the plates. 5. Replace the PBS with 10 ml of fresh ACM. Return the plates to the 37 incubator for 48 h. 6. Change the medium every 2 days until cells form a confluent monolayer. Cells usually form a confluent monolayer 10–12 days after dissection. Confluent plates contain 4 106 to 5 106 cells. 7. After 3 weeks in culture, routinely more than 95% of the cells are positively stained for glial fibrillary acidic protein (GFAP). 8. Confluent cells can then be kept for 2 weeks (changing medium twice a week) or be passed once onto poly‐L‐ornithine–coated plates or glass coverslip for further enrichment. 9. For better results, astrocytes must be passaged during the first week after confluency. 10. Wash cells in calcium‐ and magnesium‐free PBS. 11. Add 1 ml of trypsin 0.25%, EDTA 0.02% per 90‐mm tissue culture dish and incubate for 5 min at 37 . 12. Detach the cells by flushing the trypsin solution on the monolayer, add the cell suspension in a 15‐ml tube containing 14 ml of ACM and centrifuge for 8 min at 800 rpm. 13. Resuspend the cell pellet in the appropriate ACM volume, so that cell density/surface is divided three times. (One 90‐mm dish will give rise to three 90‐mm dishes of confluent astrocytes or to the equivalent surface on glass coverslips). 14. Secondary cultures are grown to confluency changing medium every 48 h and can be kept for a further 2–3 weeks changing medium twice a week. 15. We do not recommend a second passage.
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Scratch‐Induced Astrocyte Migration
Astrocytes like many other cell types will undergo migration after scratching of the cell monolayer (Fig. 1). Therefore, cell migration will occur whether you use fully differentiated astrocytes or not. However, astrocyte‐specific responses, in particular astrocyte protrusion, will occur only if the cells are fully differentiated. In vitro differentiation of astrocytes can be monitored by the nature of the intermediate filaments. Differentiated astrocytes are characterized by GFAP expression (Eliasson et al., 1999; Pekny and Pekna, 2004). Regular GFAP staining is recommended. Because the number of differentiated astrocytes increases while the cells are maintained in culture as confluent monolayers, the longer you wait the more differentiated they will be. So, we recommend waiting at least 7 days after confluence before performing a scratch assay.
FIG. 1. Scratch‐induced migration of primary rat astrocytes. Phase‐contrast images from a video showing scratch‐induced astrocyte migration. The time period after scratching is indicated at the bottom of each picture. Note the morphological changes (protrusion formation) occurring during the first 12 h and the nuclear movement only seen later. Bar, 100 m.
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Single Wound Procedure for Cell Biology Analysis 1. Use a confluent monolayer of astrocytes plated on a glass coverslip (diameter, 12–14 mm) placed in a 4‐well plate. 2. Change the cell culture medium 16 or 24 h before scratching. 3. Make a single scratch in the monolayer by moving a flamed glass capillary at regular speed across the glass coverslip. Depending on the size of the glass capillaries, the width of the scratch will vary. Because astrocytes migrate relatively slowly, scratches 300‐mm wide are sufficient for most experiments. However, for migration assays, use large glass capillaries or Pasteur pipette to make wide scratch (500 m). 4. Do not change the medium and return the coverslip in the 4‐well plate in the incubator for the desired time period. Multiple Wound Procedure for Biochemical Analysis To assess regulation of enzymatic activity (kinase assays), changes in protein content, GTP‐loading of GTPases, or protein complex formation, you must first obtained a large number of migrating astrocytes. To scale up the number of scratch‐activated cells, a large number of scratches is done on large plate of confluent cells. 1. Use confluent monolayers of astrocytes plated on 35‐mm dishes or 90‐mm dishes. 2. Change the cell culture medium 16 h before scratching. 3. Make multiple scratches in the monolayer so that at least 50% of the cells are on the edge of a scratch. We recommend the use of an eight‐channel pipet with 1–10 l tips (three tips are enough to scratch a 35‐mm dish). Slide the eight tips altogether three times around the plate and 10 times across it turning the plate a 20‐degree angle each time. 4. Incubate the plate for the required amount of time in a 37 , 5% CO2 incubator. Analysis of Scratch‐Induced Astrocyte Responses
Procedure for Immunostaining 1. Wash cells twice with 1 ml PBS. 2. Fix cells with 250 l PBS, paraformaldehyde 4%, (supplemented with glutaraldehyde 0.5% for microtubules or actin filament staining) for 10 min at room temperature.
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3. Wash twice with 1 ml PBS. 4. Permeabilize the cells with PBS, Triton 0.2%, for 10 min at room temperature. 5. Wash twice with 1 ml PBS. 6. Quench for 10 min at room temperature with 2 mg/ml NaBH4 in PBS. 7. Wash twice with 1 ml PBS. 8. Incubate with primary antibodies for 45 min at room temperature. 9. Wash 10 times in PBS. 10. Incubate with labeled‐secondary antibodies for 30 min at room temperature. 11. Wash 10 times with PBS and twice more with H2O. 12. Mount the coverslip in 5 l of Mowiol (Calbiochem) or other mounting reagent. Let the Mowiol dry for 30 min at 37 or overnight at 4 .
Astrocyte Migration Assay One must be careful not to assess cell protrusion instead of cell translocation. Indeed, the cell leading edge movement is not representative of the movement of the cell rear. Nuclear migration is barely visible during the first 6 h, and translocation of the cell body starts 8–12 h after scratching, while protrusions have already invaded the space left by the scratch (Fig. 2A). Therefore, we strongly recommend assessment of nuclear migration as a valuable sign of astrocyte migration rather than the closure of the wound, which reflects both migration and protrusion. 1. Make a large wound (approximately 500‐mm wide) across the astrocyte monolayer plated on a 12‐ to 14‐mm coverslip. 2. Immediately microinject pEGFP vector in the nucleus of cells on the edge of the scratch. 3. Place the cells back in the 37 , 5% CO2 incubator and leave them to migrate for 48 h. 4. Wash cells twice with 1 ml PBS. 5. Incubate the cells with Hoechst solution for 10 min at room temperature. Hoechst staining is sufficient to assess astrocyte migration; however, staining for actin, microtubules, or any protein of interest can be performed on the same cells. In this case, use the immunostaining procedure accordingly. 6. Wash four times in PBS. 7. Mount the cells by inverting the glass coverslip on a microscope slide on which you will have put 5 l Mowiol.
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8. Observe under the microscope at low magnification (10 or 20 objectives). Looking at the Hoechst staining, you will easily recognize the initial position of the scratch as a line with more numerous (corresponding to the accumulation of nonmigrating cells) and condensed nuclei (which mark dead cells left after scratching) (Fig. 2A, right panel). Find the GFP‐expressing cells and among these score as migrating cells those cells whose nuclei have moved away from their original place. Do not expect all expressing cells to have migrated. Approximatively only 60% of expressing cells are migrating cells. An alternative (and more fastidious) way to test astrocyte migration is to do a time‐lapse movie for 36–48 h after scratching and to assess nuclear migration by tracking the nuclei of wound‐edge cells during wound closure. Quantification of Astrocyte Protrusion We have defined astrocyte protrusion as a morphological characteristic of migrating astrocytes (Etienne‐Manneville and Hall, 2001). Protrusion starts during the first hour after scratching (Fig. 2B). However, for the first 4–6 h, the protrusion is comparable in shape and length to a fibroblast lamellipodia. It is only after 8 h that the elongation of the protrusion becomes evident. The protrusion being very flat, we recommend the use of microtubule staining (Fig. 2B). Indeed, microtubules elongate in the entire protrusion and allow easy measurement of the cell length. Protrusion formation can, therefore, be assessed 8 h after wounding; however, for easier quantification, we generally score the number of protrusions formed 16 h after scratching. Except for the migration time, the protocol is identical in both cases. 1. Make a wound (approximately 300‐mm wide) across the astrocyte monolayer plated on a 12‐ to 14‐mm coverslip.
FIG. 2. Scratch‐induced astrocyte responses. (A) Cell migration. Left panel shows tubulin (microtubules, green) and Hoechst (nuclei, blue) 24 h after scratching. From the Hoechst image shown on the right panel, the initial position of the scratch can be drawn (red line). Bar, 100 m. (B) Protrusion formation and polarization of the microtubule network. Staining with antitubulin antibodies at indicated time after scratching shows the elongation of the microtubule network and its polarization along the axis of migration. Bar, 10 m. (C) Centrosome reorientation. Concomitant Hoechst (nuclei, blue) and pericentrin (centrosome, red) staining allow the quantification of centrosome reorientation. In the left panel (0 h after scratching), the two cells of the front row are not polarized, their centrosome is not localized in the forward‐facing quadrant. In the right panel (8 h after scratching), the centrosomes of the scratch edge cells are localized in the forward‐facing quadrant. Bar, 10 m.
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2. Immediately microinject pEGFP vector or your vector of interest in the nucleus of cells of the wound edge. 3. Place the cells back in the 37 , 5% CO2 incubator and leave them to migrate for 16 h. 4. Wash cells twice with 1 ml PBS. 5. Use the immunostaining procedure with antibodies directed against tubulin. 6. Observe under the fluorescence microscope using (20 or 40 objective). Find the GFP/protein of interest expressing cells. Among these cells, looking at the microtubule staining, score as ‘‘protruding’’ cells with one major protrusion that appear at least four times longer than wide. Do not expect all expressing cells to have protrusions. Approximately 65% of GFP‐expressing cells form protrusions. We score protruding cells regardless of the orientation of the protrusion. When polarization is inhibited in the entire monolayer (for instance with PKC or GSK3 inhibitors (Etienne‐ Manneville and Hall, 2001, 2003), the number of protruding cells remains normal; however, protrusions form in random directions, and smaller protrusions can be seen on the sides of the major protrusion. Centrosome Reorientation Assay The centrosome reorientation assay allows a precise quantification of cell orientation (Fig. 2C). In astrocytes, the centrosome also serves as the major microtubule organizing center, where most microtubule minus‐ends are anchored. In a confluent astrocyte monolayer, the centrosome is localized in close proximity to the nuclear envelope in a random position around the nucleus. On scratching, the centrosome of most cells (about 75%, 16 h after scratching) localizes in front of the nucleus toward the front edge (Etienne‐Manneville and Hall, 2001). Centrosome reorientation essentially occurs during the first 6–8 h after scratching and before cell translocation. 1. For centrosome reorientation assay, allow the cells to migrate for 8 h. 2. Wash cells twice with 1 ml PBS. 3. Use the immunostaining procedure with antibodies directed against pericentrin and Hoechst. 4. Observe under the microscope using 63 objective. Centrosome reorientation must be assessed in the cells located at the wound edge
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(Fig. 2C). Eight hours after wounding, the centrosome of cells in the second and third rows of cells is not reoriented yet. To quantify centrosome reorientation, separate the space in four equal quadrants joining in the center of the nucleus of interest and placed so that one of the quadrant is facing the scratch (the median of each 90‐ degree angle being either parallel or perpendicular to the scratch) (Fig. 2C). Do not try to determine centrosome orientation along curved or irregular portions of the scratch. Centrosomes are scored as ‘‘polarized’’ or ‘‘reoriented’’ when the centrosome is placed in the quadrant facing the scratch. When cells have two centrosomes (i.e., cells in G2), centrosomes are scored polarized if one of the two centrosomes is correctly positioned. Note that in a nonpolarized population of cells, such as wound edge cells just after wounding, 25% of the centrosome will be positively scored; 25% of ‘‘polarized’’ centrosome, therefore, corresponds to a random cell orientation. Eight hours after wounding, you can expect approximately 65% of ‘‘polarized’’ centrosomes.
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