Figure 7.
Contractomeric proteins interact with actin filaments. (A) Negative-stain electron microscopy showing actin filaments buckling at the contractomere to generate an actin “ball” (also see Figs. S10 and S11). Left panel shows bright green actin puncta assembled on contractomeres (see Methods). Middle and right panels show actin filaments (arrows) being rolled into a “ball” (circled). Scale bars on two left panels are 2 μm. Scale bar on the right panel is 200 nm. (B) Negative-stain electron microscopy shows the interaction of actin filament with contractomere via multiple densities (also see Fig. S12) including a myosin-like linker extending from the membrane (red arrows, left two panels, see Materials and methods). The right panel shows an actin filament (yellow arrows) interacting with two motor heads of a myosin-like monomer (blue arrows). The tail region of a myosin-like monomer is interacting with electron-dense materials (red arrows, right panel), analogous to “stuff” associated with the tails of myosin II monomers in cells (see Results & Discussion). Scale bar on the left panel is 200 nm. Scale bar on middle and right panels are 20 nm. (C) Reconstitution actin assembly assay using stripped membranes and recombinant full-length or truncated α-actinin-4 proteins. Bottom panels show α-actinin-4 that has only one actin-binding domain cannot crosslink Alexa 647-labelled actin filaments into bundles. Scale bar is 2 μm. (D) Actin assembly on contractomeres requires α-actinin-4 binding to actin filaments. Reconstitution assay showing α-actinin-4 lacking the actin-binding domains was recruited to contractomeres but failed to support actin assembly. Left panels show membranes using phase contrast microscopy. Middle panels show targeting of α-actinin-4 to contractomeres. Right panels show actin assembly on α-actinin-4-decorated contractomeres. Graph shows intensity measurement of Oregon green-labelled α-actinin-4 on individual contractomeres; boxes represent 75 percentiles and error bars are standard deviations. Membrane-bound signals are m-ABD (n = 94), m-A4 (n = 105), m-SREF (n = 136) and contractomere signals are p-A4 (n = 93) and p-SREF (n = 64). Error bars represent standard errors. Wilcoxon–Mann–Whitney test P < 0.0001 between m-A4 and p-A4 and between m-SREF and p-SREF. P > 0.1 between p-A4 and p-SREF. Scale bars are 2 μm. (E) Contractomeric actin assembly only requires one actin-binding domain of α-actinin-4. Upper panels show membranes using phase contrast microscopy. The lower panels show actin assembly on contractomeres pre-bound with α-actinin-4. Actin assembly on contractomeres is equally robust using α-actinin-4 with one or two actin domains. Graph shows intensity measurements of rhodamine-labelled actin on individual contractomeres. Bars mark the means. m-SREF (n = 52), p-MT1ABD (n = 47) and p-MT2ABD (n = 43) refer to contractomeric signals. Wilcoxon-Mann-Whitney test P value < 0.001 between p-MT1ABD and p-SREF. P < 0.0001 between p-MT2ABD and p-SREF. P > 0.1 between p-MT1ABD and p-MT2ABD. Scale bars are 2 μm. (F) Pulse-chase actin assembly using 0.5 µM Oregon green-labeled G-actin followed by 0.5 µM rhodamine-labeled G-actin to selectively allow actin monomer addition to actin barbed-ends only (see Results & Methods). Barbed-end elongation of red G-actin shows that actin-barbed ends are most likely to be facing away from the contractomere. Scale bars are 500 nm. (G) Localization of myosin IIB, synaptopodin, α-actinin-4, and Arp2/3 at the contractomeres of native purified membranes. Upper panels show immunofluorescence of myosin IIB and synaptopodin colocalize with α-actinin-4 on contractomeres. Scale bar is 1 μm. Lower panels show myosin IIB and Arp2/3 localized to sites of actin assembly on junction membranes. Actin assembly on contractomeres is initiated by adding G-actin to purified membranes in the presence of ATP (see Materials and methods). Immunofluorescence staining for myosin IIB was performed on membranes after an actin assembly assay. Scale bar is 500 nm. (H) Actin assembly on purified native membrane is blocked by the addition of blebbistatin to the actin assembly mixture (see Methods). Scale bar is 5 μm. (I) Summary of result and working model. Contractomeric α-actinin-4 can hold onto the side of an actin filament with the filament barbed-end facing away from the contractomere. Myosin IIB can walk towards the barbed-end of the actin filament, exerting force and buckling the actin filament.

Contractomeric proteins interact with actin filaments. (A) Negative-stain electron microscopy showing actin filaments buckling at the contractomere to generate an actin “ball” (also see Figs. S10 and S11). Left panel shows bright green actin puncta assembled on contractomeres (see Methods). Middle and right panels show actin filaments (arrows) being rolled into a “ball” (circled). Scale bars on two left panels are 2 μm. Scale bar on the right panel is 200 nm. (B) Negative-stain electron microscopy shows the interaction of actin filament with contractomere via multiple densities (also see Fig. S12) including a myosin-like linker extending from the membrane (red arrows, left two panels, see Materials and methods). The right panel shows an actin filament (yellow arrows) interacting with two motor heads of a myosin-like monomer (blue arrows). The tail region of a myosin-like monomer is interacting with electron-dense materials (red arrows, right panel), analogous to “stuff” associated with the tails of myosin II monomers in cells (see Results & Discussion). Scale bar on the left panel is 200 nm. Scale bar on middle and right panels are 20 nm. (C) Reconstitution actin assembly assay using stripped membranes and recombinant full-length or truncated α-actinin-4 proteins. Bottom panels show α-actinin-4 that has only one actin-binding domain cannot crosslink Alexa 647-labelled actin filaments into bundles. Scale bar is 2 μm. (D) Actin assembly on contractomeres requires α-actinin-4 binding to actin filaments. Reconstitution assay showing α-actinin-4 lacking the actin-binding domains was recruited to contractomeres but failed to support actin assembly. Left panels show membranes using phase contrast microscopy. Middle panels show targeting of α-actinin-4 to contractomeres. Right panels show actin assembly on α-actinin-4-decorated contractomeres. Graph shows intensity measurement of Oregon green-labelled α-actinin-4 on individual contractomeres; boxes represent 75 percentiles and error bars are standard deviations. Membrane-bound signals are m-ABD (n = 94), m-A4 (n = 105), m-SREF (n = 136) and contractomere signals are p-A4 (n = 93) and p-SREF (n = 64). Error bars represent standard errors. Wilcoxon–Mann–Whitney test P < 0.0001 between m-A4 and p-A4 and between m-SREF and p-SREF. P > 0.1 between p-A4 and p-SREF. Scale bars are 2 μm. (E) Contractomeric actin assembly only requires one actin-binding domain of α-actinin-4. Upper panels show membranes using phase contrast microscopy. The lower panels show actin assembly on contractomeres pre-bound with α-actinin-4. Actin assembly on contractomeres is equally robust using α-actinin-4 with one or two actin domains. Graph shows intensity measurements of rhodamine-labelled actin on individual contractomeres. Bars mark the means. m-SREF (n = 52), p-MT1ABD (n = 47) and p-MT2ABD (n = 43) refer to contractomeric signals. Wilcoxon-Mann-Whitney test P value < 0.001 between p-MT1ABD and p-SREF. P < 0.0001 between p-MT2ABD and p-SREF. P > 0.1 between p-MT1ABD and p-MT2ABD. Scale bars are 2 μm. (F) Pulse-chase actin assembly using 0.5 µM Oregon green-labeled G-actin followed by 0.5 µM rhodamine-labeled G-actin to selectively allow actin monomer addition to actin barbed-ends only (see Results & Methods). Barbed-end elongation of red G-actin shows that actin-barbed ends are most likely to be facing away from the contractomere. Scale bars are 500 nm. (G) Localization of myosin IIB, synaptopodin, α-actinin-4, and Arp2/3 at the contractomeres of native purified membranes. Upper panels show immunofluorescence of myosin IIB and synaptopodin colocalize with α-actinin-4 on contractomeres. Scale bar is 1 μm. Lower panels show myosin IIB and Arp2/3 localized to sites of actin assembly on junction membranes. Actin assembly on contractomeres is initiated by adding G-actin to purified membranes in the presence of ATP (see Materials and methods). Immunofluorescence staining for myosin IIB was performed on membranes after an actin assembly assay. Scale bar is 500 nm. (H) Actin assembly on purified native membrane is blocked by the addition of blebbistatin to the actin assembly mixture (see Methods). Scale bar is 5 μm. (I) Summary of result and working model. Contractomeric α-actinin-4 can hold onto the side of an actin filament with the filament barbed-end facing away from the contractomere. Myosin IIB can walk towards the barbed-end of the actin filament, exerting force and buckling the actin filament.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal