Myosin-II negatively regulates minor process extension and the temporal development of neuronal polarity

Abstract

The earliest stage in the development of neuronal polarity is characterized by extension of undifferentiated "minor processes" (MPs), which subsequently differentiate into the axon and dendrites. We investigated the role of the myosin II motor protein in MP extension using forebrain and hippocampal neuron cultures. Chronic treatment of neurons with the myosin II ATPase inhibitor blebbistatin increased MP length, which was also seen in myosin IIB knockouts. Through live-cell imaging, we demonstrate that myosin II inhibition triggers rapid minor process extension to a maximum length range. Myosin II activity is determined by phosphorylation of its regulatory light chains (rMLC) and mediated by myosin light chain kinase (MLCK) or RhoA-kinase (ROCK). Pharmacological inhibition of MLCK or ROCK increased MP length moderately, with combined inhibition of these kinases resulting in an additive increase in MP length similar to the effect of direct inhibition of myosin II. Selective inhibition of RhoA signaling upstream of ROCK, with cell-permeable C3 transferase, increased both the length and number of MPs. To determine whether myosin II affected development of neuronal polarity, MP differentiation was examined in cultures treated with direct or indirect myosin II inhibitors. Significantly, inhibition of myosin II, MLCK, or ROCK accelerated the development of neuronal polarity. Increased myosin II activity, through constitutively active MLCK or RhoA, decreased both the length and number of MPs and, consequently, delayed or abolished the development of neuronal polarity. Together, these data indicate that myosin II negatively regulates MP extension, and the developmental time course for axonogenesis.

Figure 1

Myosin IIA and IIB heavy chain isoforms are differentially expressed in embryonic neurons during early developmental stages. (A–B) Subcellular distribution of myosin II isoforms in stage II chick forebrain neurons at 2 DIV, immunostained for α-tubulin (green) and myosin IIA or myosin IIB heavy chains (red) using isoform-specific polyclonal antibodies. Selected minor processes depicted as enlarged insets. (A) Minor processes stain robustly for myosin IIA, distributed as small puncta throughout their length and often enriched within tips characteristic of elaborated growth cones. (B) In contrast, myosin IIB staining is uniform but faint within minor processes. (C) Western blot of E8 chick forebrain extract prepared from 1–1.5 DIV cultures showing relative levels of myosin IIA (lane 1) and IIB (lane 2) heavy chain isoform proteins. (D–E) Subcellular distribution of myosin II isoforms in stage II rat hippocampal neurons at 3 DIV, immunostained for α-tubulin (green) and myosin IIA or myosin IIB heavy chains (red). Selected minor processes depicted as enlarged insets. Myosin IIB staining predominates in developing minor processes (E), while myosin IIA levels are barely detectible (D).

Figure 2

Inhibition of myosin II activity increases process length but decreases process number. (A) Forebrain neuron cultures were treated with 50 µM blebbistatin or DMSO vehicle control for 2 DIV, then fixed and immunostained for α-tubulin (green) and F-actin (red) to reveal minor processes. (B) Quantification of minor process length showed an increase with blebbistatin treatment (n = 289 processes), relative to DMSO controls (n = 297; mean = 19 ± 0.5 µm). (C) Still frames from phase-contrast live-cell imaging of stage II forebrain neurons immediately before (time 0) and one hour after (time 60) treatment with blebbistatin or DMSO vehicle. Minor processes exposed to blebbistatin extended rapidly and steadily (arrowheads indicate position of growth cones prior to treatment) while DMSO treated processes exhibited no net growth. (D) Quantification of minor process number showed a reduction with blebbistatin treatment (n = 304 neurons) as compared to DMSO controls (n = 344; mean = 6.5 ± 0.1). (E) Rat hippocampal neuron cultures were treated with 50 µM blebbistatin or DMSO vehicle control for 2 DIV, then fixed and immunostained for α-tubulin (green) and F-actin (red) to reveal neurites. (F) Quantification of murine neurite length showed an increase with blebbistatin exposure (n = 20 neurons), knockout of myosin IIB gene expression (n = 24), or blebbistatin treatment of myosin IIB knockouts (n = 24), relative to wild type DMSO controls (n = 38; mean = 123.8 ± 9.7 µm). Data are presented normalized to control values. **p ≤ 0.01, ***p ≤ 0.0001, ****p ≤ 0.00001, Welch t-test. Error bars indicate SEM.

Figure 3

Combined MLCK and ROCK activity regulates minor process growth. (A) Forebrain neurons fixed at 2 DIV and immunostained for MLCK (green) and phalloidin-stained for F-actin (red). MLCK staining is evident within the soma and throughout minor processes of stage II neurons, often appearing enriched in areas also enriched for F-actin. (B) Forebrain neurons fixed at 2 DIV and immunostained for ROCK (green) and phalloidin-stained for F-actin (red). ROCK levels are highest within the soma and more diffuse within minor processes, also appearing to co-localize with regions of high F-actin content. (C) Quantification of minor process length following inhibition of MLCK or ROCK. Forebrain neurons were maintained for 2 DIV in the presence of MLCK inhibitor (ML-7, 500 nM), Rho Kinase inhibitor (Y-27632, 10 µM), both ML-7 and Y-27632, or DMSO vehicle. Chronic treatment with ML-7 (n = 622 processes) or Y-27632 (n = 526) increased minor process length relative to DMSO controls (n = 586), while combined treatment with ML-7 and Y-27632 (M+Y, n = 554) produced an increase in length similar to that produced with blebbistatin treatment. (D–E) Forebrain neuron cultures at 2 DIV were treated with the RhoA inhibitor, cell-permeable C3-transferase, for 4 hours prior to fixation. Both the length (D) and number (E) of minor processes were increased following exposure to C-3 transferase (n = 1385 processes, 369 neurons), relative to mock-treated controls (n =1697 processes, mean = 23.8 ± 0.2 µm; n = 343 neurons, mean = 9.2 ± 0.15 processes). Data are presented normalized to the control values. ****p ≤ 0.00001, Welch t-test. Error bars indicate SEM.

Figure 4

Both MLCK and ROCK partially regulate myosin II activity through myosin light chain phosphorylation. (A) Immunodetection of phosphorylated regulatory myosin light chains (rMLC-p). Forebrain neurons were maintained for 2 DIV in the presence of MLCK inhibitor (ML-7, 500 nM), Rho Kinase inhibitor (Y-27632, 10 µM), or DMSO vehicle control. Neurons were loaded with 2.5 µM of the volumetric fluorescent reporter CellTracker, then fixed and immunostained with an antibody to rMLC-p (green) and phalloidin-stained for F-actin (red) to reveal minor processes. Relative to the DMSO mean rMLC-p staining intensity, both ML-7 and Y-27632 reduced staining within minor processes. (B) Quantification of rMLC-p staining intensity was performed using previously published ratiometric methods (Louden et al., 2006), and revealed partial reduction in rMLC-p staining with ML-7 (n = 168 processes) or Y-27632 (n = 136) as compared to DMSO (n = 136). Data are presented normalized to the DMSO control values. ****p ≤ 0.00001, Welch t-test. Error bars indicate SEM.

Figure 5

Myosin II inhibition produces stable increases in minor process length. (A) Chick metanephric cell cultures were treated with blebbistatin (50 µM) for 24 or 48 hours and fixed, or treated with blebbistatin for 24 hours followed by washout for another 24 hours prior to fixation. Blebbistatin-treated cells displayed a loss of stress fibers with staining for α-tubulin (green) and F-actin (red), which was reversed following washout. (B) Blebbistatin washout was performed in forebrain neuron cultures as described for metanephric cultures, and minor process lengths were quantified (C). Minor process lengths were similar in cultures treated with blebbistatin for 24 hours and fixed at 1 DIV (n = 779 processes), or fixed at 2 DIV following washout (n = 1059), which were greater than control neuron lengths at 1 DIV (n = 789) or 2 DIV (n = 1015). The greatest lengths were attained by cultures under blebbistatin treatment for 48 hours (n = 791). Data are presented normalized to the DMSO control at 24 hours. ****p ≤ 0.00001, NS = non-significant, Welch t-test. Error bars indicate SEM.

Figure 6

Myosin II inhibition alters minor process growth dynamics. The effects of acute or chronic blebbistatin (50 µM) on minor process dynamics were determined through live cell imaging of forebrain neurons cultured for 30 minutes, 1 DIV, or 2 DIV. Acute blebbistatin treatment produced the greatest proportion of growing processes at each time point (30 minutes, n = 52 processes; 1 DIV, n = 85; 48 hours, n = 66), relative to chronic blebbistatin exposure or DMSO vehicle. Within DMSO control cultures at each time point, nearly equal numbers of processes were seen undergoing periods of growth or retraction (30 minutes, n = 71 processes; 1 DIV, n = 128; 2 DIV, n = 67). Chronic blebbistatin treatment (1 DIV, n = 141; 2 DIV, n = 40) resulted in a higher proportion of processes extending than in DMSO controls, though not as high as the proportion in acute-blebbistatin treated cultures. Data are presented as the percentage of minor processes growing (bars above the origin) or retracting (bars below the origin). *p ≤ 0.01, ***p ≤ 0.0001, Welch t-test. Error bars indicate SEM.

Figure 7

Myosin II inhibition rapidly increases minor process lengthening. (A) The effects of acute or chronic blebbistatin (50 µM) treatment on minor process dynamics were examined through live cell imaging of forebrain neurons cultured for 30 minutes, 1 DIV, or 2 DIV. The change in minor process length between the first (time 0) and last (time 60) frame of each hour long experiment was quantified. At each developmental time point, acute blebbistatin treatment (30 min., n = 52 processes; 1 DIV, n = 85; 2 DIV n = 66) increased minor process length as compared to DMSO controls (30 min., n = 71; 1 DIV, n = 128; 2 DIV n = 67). Chronic blebbistatin treatment (1 DIV, n = 141; 2 DIV n = 40) produced lower magnitude increases in process length than acute treatment. (B) Normalization of minor process growth reveals that acute blebbistatin treatment produced a similar enhancement of growth at each developmental time point, while DMSO control processes exhibited no net growth. Following chronic blebbistatin treatment, increases in process growth were only seen at 1 DIV. Sample sizes indicated above. Data are presented as the percentage of the initial length. *p ≤ 0.05, ****p ≤ 0.00001, Welch t-test. Error bars indicate SEM.

Figure 8

Inhibition of myosin II activity accelerates the development of neuronal polarity. (A–B) Mouse hippocampal cultures were maintained for 3 DIV in the presence of DMSO vehicle or blebbistatin, respectively, then fixed and immunostained to reveal the distribution of tyrosinated tubulin (green) and tau-1 (red), an axonal marker. Axon specification is not affected by blebbistatin treatment. Arrows indicate tau-1 positive axons. Note the absence of tau-1 staining in dendrites (asterisks). (C–D) Rat hippocampal cultures were maintained for 3 DIV in the presence of DMSO vehicle or blebbistatin, respectively, then fixed and immunostained to reveal the distribution of tau-1 (red), and dendrite-specific MAP-2 (green), with overlap shown as yellow. Arrows indicate single tau-1 positive axons. (E) Forebrain neurons were maintained for 2 DIV in the presence of blebbistatin (50 µM), ML-7 (500 nM), Y-27632 (10 µM), both ML-7 and Y-27632, or DMSO, and the proportion of neurons at each developmental stage was determined for each treatment population. The distribution of neurons was shifted toward more mature morphological stages by blebbistatin (n = 19 fields), and to a lesser extent by ML-7 (n = 16), Y-27632 (n = 24), or a combination of these drugs (n = 18), as compared to DMSO controls (n = 12). (F) Hippocampal neurons were cultured in the presence of blebbistatin (n = 15 fields per time point) or DMSO (n = 15) and fixed at 1, 2, and 3 DIV to determine the proportion of neurons at each developmental stage. Similar to forebrain cultures, the distribution of neurons was shifted toward more mature morphological stages by blebbistatin relative to DMSO. Data are presented as the percentage distribution. *p ≤ 0.05, **p ≤ 0.001, ***p ≤ 0.0001, ****p ≤ 0.00001, Welch t-test. Error bars indicate SEM.

Figure 9

Increased myosin II activity restricts minor process extension. (A) forebrain neurons were transfected with EGFP cDNA, or EYFP-tagged cDNA constructs encoding wild type myosin light chain kinase (WT-MLCK) or constitutively active MLCK (CA-MLCK), then fixed at 2 DIV and immunostained for EGFP/EYFP (green) to reveal minor processes. (B–C) Minor process length and number were decreased in stage II forebrain neurons expressing CA-MLCK (n = 228 processes, 53 neurons) as compared to wild type controls (n = 304 processes; n = 52 neurons). (D) forebrain neuron cultures were peptide-transfected at 2 DIV with constitutively active RhoA protein (L63RhoA) or inert BSA control protein for 8 hours prior to fixation and immunostaining for the His-tag reporter, or α-tubulin (green) and F-actin (red). (E–F) Minor process length and number were decreased in stage II forebrain neurons following transfection with L63RhoA (n = 569 processes, 111 neurons) as compared to BSA peptide-transfected controls (n = 887 processes, 129 neurons). Data are presented normalized to the corresponding WT-MLCK or BSA control values. ****p ≤ 0.00001, Welch t-test. Error bars indicate SEM.

Figure 10

Increased myosin II activity inhibits the development of neuronal polarity. Quantification of morphological subtypes in forebrain neurons electroporated with control EGFP cDNA or EYFP-tagged constructs encoding wild type myosin light chain kinase (WT-MLCK) or constitutively active MLCK (CA-MLCK). Cultures were fixed at 2 DIV and immunostained for EGFP/EYFP (similarly detected with anti-EGFP antibody) to reveal morphology. All transfected neurons were scored as Stage 0, I, II, or III, and the proportion at each developmental stage was determined for each transfectant population. Development of polarity was delayed or inhibited in neuronal populations expressing CA-MLCK (n = 138 neurons) relative to both WT-MLCK (n = 393) and EGFP (n = 300) expressing populations. Data are presented as the percentage distribution. *p ≤ 0.05, ****p ≤ 0.00001 as compared to EGFP control, Welch t-test. Error bars indicate SEM.

Figure 11

Role of microtubule dynamics and actin filaments in minor process extension. (A) Microtubule dynamics are necessary for minor process length increases following myosin II inhibition. Hippocampal neurons were maintained for 2 DIV and treated with blebbistatin (100 µM), stabilizing concentrations of nocodazole (150 nM), or both blebbistatin and nocodazole for 24 hours prior to fixation. Blebbistatin treatment (n = 12 processes) produced an increase in the length of neurites over controls (n = 11, mean = 137.1 ± 25.65 µm), which was dampened by concurrent nocodazole treatment (n = 13). Nocodazole treatment alone (n = 15) diminished neurite lengths relative to controls. Data are presented normalized to the DMSO control at 24 hours. (B) F-actin disruption promotes minor process length. Forebrain neuron cultures were treated 30 minutes after plating with 50 µM blebbistatin, F-actin-depolymerizing concentrations of latrunculin A (4 µM), latrunculin plus blebbistatin, or DMSO vehicle control for 2 DIV, then fixed and immunostained for α-tubulin to reveal minor processes. Deplymerization of F-actin with latruculin (n = 560 processes) increased minor process lengths as compared to DMSO controls (n = 709), which were further increased with concurrent blebbistatin treatment. Latrunculin dampened the increase in minor process extension produced by blebbistatin treatment alone (n = 1068). **p ≤ 0.01, ***p ≤ 0.0001, Welch t-test, with paired comparisons. Error bars indicate SEM.

Figure 12

Model of the proposed mechanism for myosin II regulation during the development of neuronal polarity. In the newly post-mitotic neuron, actomyosin-based contractility maintains cellular integrity and constrains cytoskeletal dynamics. (A) Local signaling leading to changes in F-actin stability or decreases in myosin II activity trigger symmetry breaking and filopodial extension from the neuronal sphere, which may be potentiated through decreases in myosin II-mediated kinking and severing of actin bundles. (B) Ongoing myosin II activity regulates the time course for minor process growth, until an intrinsically determined maximum length is attained, (C) after which as yet uncharacterized cytoskeletal changes trigger symmetry breaking events that underlie axonal specification.