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Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.

Nishimura Y, Applegate K, Davidson MW, Danuser G, Waterman CM - PLoS ONE (2012)

Bottom Line: MARK2 was the only protein whose RNAi targeting additionally suppressed Rac1 effects on MT orientation in lamellipodia, and thus became the focus of further study.We show that GFP-MARK2 rescued effects of MARK2 depletion on MT growth lifetime and orientation, and GFP-MARK2 localized in lamellipodia in a Rac1-activity-dependent manner.In a wound-edge motility assay, MARK2-depleted cells failed to polarize their centrosomes or exhibit oriented MT growth in the leading edge, and displayed defects in directional cell migration.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Polarized microtubule (MT) growth in the leading edge is critical to directed cell migration, and is mediated by Rac1 GTPase. To find downstream targets of Rac1 that affect MT assembly dynamics, we performed an RNAi screen of 23 MT binding and regulatory factors and identified RNAi treatments that suppressed changes in MT dynamics induced by constitutively activated Rac1. By analyzing fluorescent EB3 dynamics with automated tracking, we found that RNAi treatments targeting p150(glued), APC2, spastin, EB1, Op18, or MARK2 blocked Rac1-mediated MT growth in lamellipodia. MARK2 was the only protein whose RNAi targeting additionally suppressed Rac1 effects on MT orientation in lamellipodia, and thus became the focus of further study. We show that GFP-MARK2 rescued effects of MARK2 depletion on MT growth lifetime and orientation, and GFP-MARK2 localized in lamellipodia in a Rac1-activity-dependent manner. In a wound-edge motility assay, MARK2-depleted cells failed to polarize their centrosomes or exhibit oriented MT growth in the leading edge, and displayed defects in directional cell migration. Thus, automated image analysis of MT assembly dynamics identified MARK2 as a target regulated downstream of Rac1 that promotes oriented MT growth in the leading edge to mediate directed cell migration.

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Effects of MT regulatory protein depletion on MT growth dynamics in U2-OS cells.Top: Results of analysis of time-lapse movies of mKO-EB3 with PlusTipTracker software. Proportion of MT growth excursions in each subpopulation in control shRNA vector-transfected cells (WT) or cells treated with RNAis targeting the protein noted (kd). shRNA vectors were used for RNAi targeting of EB1, CLASP2, dynamitin, DCX, MAP1A, MAP1B, MAP2, MAP4, MARK1, MARK2 and MARK3. siRNA oligos were used for RNAi targeting of APC, APC2, ACF7, XMAP215, Op18, p150glued, CLIP115, CLIP170, STOP, MAP1S, Spastin and Katanin p60 (see Methods). Bottom: Color key showing MT growth speed and growth excursion lifetime ranges for each subpopulation.
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pone-0041413-g002: Effects of MT regulatory protein depletion on MT growth dynamics in U2-OS cells.Top: Results of analysis of time-lapse movies of mKO-EB3 with PlusTipTracker software. Proportion of MT growth excursions in each subpopulation in control shRNA vector-transfected cells (WT) or cells treated with RNAis targeting the protein noted (kd). shRNA vectors were used for RNAi targeting of EB1, CLASP2, dynamitin, DCX, MAP1A, MAP1B, MAP2, MAP4, MARK1, MARK2 and MARK3. siRNA oligos were used for RNAi targeting of APC, APC2, ACF7, XMAP215, Op18, p150glued, CLIP115, CLIP170, STOP, MAP1S, Spastin and Katanin p60 (see Methods). Bottom: Color key showing MT growth speed and growth excursion lifetime ranges for each subpopulation.

Mentions: To find downstream targets of Rac1 that promote pioneer MTs, we performed RNAi-based depletion of 23 MT-regulatory factors (Figs. 2 and 3) and screened for proteins whose depletion suppresses the slow, short-lived MT growth parallel to the leading edge induced by CA-Rac1 in U2-OS cells (Fig. 3). We focused our screen on non-motor MT-binding target proteins that are known to regulate MT growth behavior, but it is unknown if they are regulated by Rac1. These included APC, APC2, ACF7, CLASP2, XMAP215, CLIP170/115, p150glued, p50/dynamitin, doublecortin, EB1, katanin p60 subunit, MARK1, MARK2, MARK3, MAP1A, MAP1B, MAP1S, MAP2, MAP4, STOP, spastin, or Op18/stathmin. shRNA vectors were used for RNAi targeting of EB1, CLASP2, dynamitin, DCX, MAP1A, MAP1B, MAP2, MAP4, MARK1, MARK2 and MARK3. siRNA oligos were used for RNAi targeting of APC, APC2, ACF7, XMAP215, Op18, p150glued, CLIP115, CLIP170, STOP, MAP1S, spastin and katanin p60 (see Methods). Op18/stathmin and CLASP that are both known to regulate MT dynamics downstream of Rac1 in other cell types [13], [15] were included as positive controls. We used plusTipTracker to analyze the speed, lifetime, and orientation of MT growth excursions in movies of cells expressing mKO-EB3 and treated with either siRNAs or shRNAs targeting (see Methods) one of these MT regulatory proteins together with (Fig. 3A, Tables S5, S6) or without (Fig. 2, Tables S3, S4) the additional expression of CA-Rac1. Expression of target proteins in U2OS cells was verified for as many target proteins as possible (see methods), however in many cases antibodies were not available. Thus, negative results in this screen should be interpreted with caution.


Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells.

Nishimura Y, Applegate K, Davidson MW, Danuser G, Waterman CM - PLoS ONE (2012)

Effects of MT regulatory protein depletion on MT growth dynamics in U2-OS cells.Top: Results of analysis of time-lapse movies of mKO-EB3 with PlusTipTracker software. Proportion of MT growth excursions in each subpopulation in control shRNA vector-transfected cells (WT) or cells treated with RNAis targeting the protein noted (kd). shRNA vectors were used for RNAi targeting of EB1, CLASP2, dynamitin, DCX, MAP1A, MAP1B, MAP2, MAP4, MARK1, MARK2 and MARK3. siRNA oligos were used for RNAi targeting of APC, APC2, ACF7, XMAP215, Op18, p150glued, CLIP115, CLIP170, STOP, MAP1S, Spastin and Katanin p60 (see Methods). Bottom: Color key showing MT growth speed and growth excursion lifetime ranges for each subpopulation.
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pone-0041413-g002: Effects of MT regulatory protein depletion on MT growth dynamics in U2-OS cells.Top: Results of analysis of time-lapse movies of mKO-EB3 with PlusTipTracker software. Proportion of MT growth excursions in each subpopulation in control shRNA vector-transfected cells (WT) or cells treated with RNAis targeting the protein noted (kd). shRNA vectors were used for RNAi targeting of EB1, CLASP2, dynamitin, DCX, MAP1A, MAP1B, MAP2, MAP4, MARK1, MARK2 and MARK3. siRNA oligos were used for RNAi targeting of APC, APC2, ACF7, XMAP215, Op18, p150glued, CLIP115, CLIP170, STOP, MAP1S, Spastin and Katanin p60 (see Methods). Bottom: Color key showing MT growth speed and growth excursion lifetime ranges for each subpopulation.
Mentions: To find downstream targets of Rac1 that promote pioneer MTs, we performed RNAi-based depletion of 23 MT-regulatory factors (Figs. 2 and 3) and screened for proteins whose depletion suppresses the slow, short-lived MT growth parallel to the leading edge induced by CA-Rac1 in U2-OS cells (Fig. 3). We focused our screen on non-motor MT-binding target proteins that are known to regulate MT growth behavior, but it is unknown if they are regulated by Rac1. These included APC, APC2, ACF7, CLASP2, XMAP215, CLIP170/115, p150glued, p50/dynamitin, doublecortin, EB1, katanin p60 subunit, MARK1, MARK2, MARK3, MAP1A, MAP1B, MAP1S, MAP2, MAP4, STOP, spastin, or Op18/stathmin. shRNA vectors were used for RNAi targeting of EB1, CLASP2, dynamitin, DCX, MAP1A, MAP1B, MAP2, MAP4, MARK1, MARK2 and MARK3. siRNA oligos were used for RNAi targeting of APC, APC2, ACF7, XMAP215, Op18, p150glued, CLIP115, CLIP170, STOP, MAP1S, spastin and katanin p60 (see Methods). Op18/stathmin and CLASP that are both known to regulate MT dynamics downstream of Rac1 in other cell types [13], [15] were included as positive controls. We used plusTipTracker to analyze the speed, lifetime, and orientation of MT growth excursions in movies of cells expressing mKO-EB3 and treated with either siRNAs or shRNAs targeting (see Methods) one of these MT regulatory proteins together with (Fig. 3A, Tables S5, S6) or without (Fig. 2, Tables S3, S4) the additional expression of CA-Rac1. Expression of target proteins in U2OS cells was verified for as many target proteins as possible (see methods), however in many cases antibodies were not available. Thus, negative results in this screen should be interpreted with caution.

Bottom Line: MARK2 was the only protein whose RNAi targeting additionally suppressed Rac1 effects on MT orientation in lamellipodia, and thus became the focus of further study.We show that GFP-MARK2 rescued effects of MARK2 depletion on MT growth lifetime and orientation, and GFP-MARK2 localized in lamellipodia in a Rac1-activity-dependent manner.In a wound-edge motility assay, MARK2-depleted cells failed to polarize their centrosomes or exhibit oriented MT growth in the leading edge, and displayed defects in directional cell migration.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
Polarized microtubule (MT) growth in the leading edge is critical to directed cell migration, and is mediated by Rac1 GTPase. To find downstream targets of Rac1 that affect MT assembly dynamics, we performed an RNAi screen of 23 MT binding and regulatory factors and identified RNAi treatments that suppressed changes in MT dynamics induced by constitutively activated Rac1. By analyzing fluorescent EB3 dynamics with automated tracking, we found that RNAi treatments targeting p150(glued), APC2, spastin, EB1, Op18, or MARK2 blocked Rac1-mediated MT growth in lamellipodia. MARK2 was the only protein whose RNAi targeting additionally suppressed Rac1 effects on MT orientation in lamellipodia, and thus became the focus of further study. We show that GFP-MARK2 rescued effects of MARK2 depletion on MT growth lifetime and orientation, and GFP-MARK2 localized in lamellipodia in a Rac1-activity-dependent manner. In a wound-edge motility assay, MARK2-depleted cells failed to polarize their centrosomes or exhibit oriented MT growth in the leading edge, and displayed defects in directional cell migration. Thus, automated image analysis of MT assembly dynamics identified MARK2 as a target regulated downstream of Rac1 that promotes oriented MT growth in the leading edge to mediate directed cell migration.

Show MeSH
Related in: MedlinePlus