Figure 5.
Manipulation of actomyosin-II activity affects the flux-induced axon beading process. (A) Lifeact-G/RFP expressing neurons were either untreated (Ctrl) or pretreated for 30 min with either Blebbistatin (+BLB; 50 μΜ), ML-7 (+ML-7; 10 μΜ), Calyculin A (+CA; 50 nM), or latrunculin B (+LatB; 5 μΜ), prior to being stressed with 50 μl/min flux for 180 s. Representative time-lapse images show the deformation of the same axons before (top) and during (bottom) the flux for each condition, with beading regions shown in magenta. Bar = 5 μm. (B and C) Quantification of (B) axon bead number and (C) recovery rate of the axon beading process induced by 50 µl/min flux for 180 s (N = 41, 27, 49, 27, 23 axons). (D) IF staining of p-MRLC after 50 nM Calyculin A treatment (+CA) for 30 min. Bar = 10 μm. (E) Quantification of axon beading density induced by CA treatment (N = 16, 19). (F) Representative 3D-SIM images depict the relative localization between βII-Spectrin (green) and p-MRLC (magenta). Bottom: Line profiles illustrate fluorescence intensity along bracketed axonal regions in control and CA-treated axons. Bar = 0.5 μm. (G) Quantification of p-MRLC periodicity in control (Ctrl) and CA-treated axons (N = 17, 17, 33). (H) Representative 3D-SIM images display the periodicity and fluorescence intensity of p-MRLC in non-fluxed (top) and fluxed axons (bottom). Non-fluxed (1#, 2#), beading (3#), and between (4#) regions marked with brackets and amplified in right panels. Red brackets in 3# indicate gaps lacking periodic p-MRLC. Bar = 1 μm. (I) Quantification of p-MRLC periodicity (N = 18, 43, 87). (J) Quantification of spacing between p-MRLC rings (N = 112, 282, 804). (K) Quantification of p-MRLC fluorescence intensity (N = 11, 13, 13). (L) Representative time-lapse images showing axons infected with AAV virus expressing either MRLC-WT, an inactive MRLC mutant (MRLC-SA), a constitutively active MRLC mutant (MRLC-SD), or an empty vector (GFP), with the deformation of the same axon induced by 50 μl/min flux shown below the corresponding condition. Bar = 10 μm. (M) Quantification of the dynamic beading process induced by a 50 µl/min flux in L. Data are presented as mean ± SEM, analyzed using an unpaired two-tailed Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001.

Manipulation of actomyosin-II activity affects the flux-induced axon beading process. (A) Lifeact-G/RFP expressing neurons were either untreated (Ctrl) or pretreated for 30 min with either Blebbistatin (+BLB; 50 μΜ), ML-7 (+ML-7; 10 μΜ), Calyculin A (+CA; 50 nM), or latrunculin B (+LatB; 5 μΜ), prior to being stressed with 50 μl/min flux for 180 s. Representative time-lapse images show the deformation of the same axons before (top) and during (bottom) the flux for each condition, with beading regions shown in magenta. Bar = 5 μm. (B and C) Quantification of (B) axon bead number and (C) recovery rate of the axon beading process induced by 50 µl/min flux for 180 s (N = 41, 27, 49, 27, 23 axons). (D) IF staining of p-MRLC after 50 nM Calyculin A treatment (+CA) for 30 min. Bar = 10 μm. (E) Quantification of axon beading density induced by CA treatment (N = 16, 19). (F) Representative 3D-SIM images depict the relative localization between βII-Spectrin (green) and p-MRLC (magenta). Bottom: Line profiles illustrate fluorescence intensity along bracketed axonal regions in control and CA-treated axons. Bar = 0.5 μm. (G) Quantification of p-MRLC periodicity in control (Ctrl) and CA-treated axons (N = 17, 17, 33). (H) Representative 3D-SIM images display the periodicity and fluorescence intensity of p-MRLC in non-fluxed (top) and fluxed axons (bottom). Non-fluxed (1#, 2#), beading (3#), and between (4#) regions marked with brackets and amplified in right panels. Red brackets in 3# indicate gaps lacking periodic p-MRLC. Bar = 1 μm. (I) Quantification of p-MRLC periodicity (N = 18, 43, 87). (J) Quantification of spacing between p-MRLC rings (N = 112, 282, 804). (K) Quantification of p-MRLC fluorescence intensity (N = 11, 13, 13). (L) Representative time-lapse images showing axons infected with AAV virus expressing either MRLC-WT, an inactive MRLC mutant (MRLC-SA), a constitutively active MRLC mutant (MRLC-SD), or an empty vector (GFP), with the deformation of the same axon induced by 50 μl/min flux shown below the corresponding condition. Bar = 10 μm. (M) Quantification of the dynamic beading process induced by a 50 µl/min flux in L. Data are presented as mean ± SEM, analyzed using an unpaired two-tailed Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal