Research ArticleVASCULAR BIOLOGY

Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility

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Sci. Signal.  19 Sep 2017:
Vol. 10, Issue 497, eaan2694
DOI: 10.1126/scisignal.aan2694
  • Fig. 1 Intact microtubules maintain close proximity of the peripheral SR and the plasma membrane.

    (A) Representative image of an isolated smooth muscle cell loaded with ER-Tracker (green) to stain the SR membrane, a cell loaded with CellMask Deep Red (red) to stain the plasma membrane (PM), and a merged image (yellow). Scale bar, 5 μm. Right: Cross-section (i and ii) images of SR (green) and plasma membrane (red) staining. Scale bar, 1 μm (middle). The regions of interest (ROIs) (box) show putative peripheral coupling sites. Enlarged images are shown below. Scale bar, 0.3 μm (bottom). (B) Line scan images showing plasma membrane–SR separation distance as a function of time in untreated control cells (n = 7 cells, n = 3 animals), cells treated with swinholide A and latrunculin B (L+S) to depolymerize actin (n = 6 cells, n = 3 animals), and cells treated with nocodazole (Noco) to depolymerize microtubules (n = 7 cells, n = 3 animals). (C) Summary data showing the relative separation distance between the SR and the plasma membrane over time. *P ≤ 0.05 compared to control and L+S.

  • Fig. 2 Arching microtubule structures underlie the peripheral SR.

    (A) 3D reconstruction of the microtubule (MT) cytoskeleton in an isolated cerebral arterial myocyte loaded with Tubulin Tracker (red) (n = 8 cells, n = 3 animals). Scale bar, 5 μm. Examples of arching microtubule structures are indicated by white arrowheads. (B) Representative compressed z-stack images (0.25 μm per slice) of isolated smooth muscle cells loaded with Tubulin Tracker (red) and ER-Tracker (green) (n = 8 cells, n = 3 animals). Scale bar, 5 μm. (C) A 3D reconstruction analysis was performed on ROIs (i) and (ii) (9.2 μm × 9.2 μm × 4.75 μm). White arrowheads indicate microtubule arches underlying the SR proximal to the plasma membrane.

  • Fig. 3 RyR2 protein clusters selectively align with microtubules.

    (A) Representative images (of five cells from n = 3 animals) of an isolated native cerebral arterial myocyte immunolabeled with anti–α-tubulin (red). The image on the left is a wide-field image. The ROI in the yellow box was imaged using GSDIM. Scale bar, 10 μm. Center: Superresolution image of the ROI. Scale bar, 3 μm. Magnified views of the indicated ROIs depicting arching microtubule structures are shown on the right. Scale bar, 0.2 μm. (B) Representative superresolution images (of five cells from n = 3 animals) of an isolated native cerebral arterial myocyte immunolabeled with anti–α-tubulin antibody (red), anti-RyR2 antibody (green), and the overlay. Scale bar, 3 μm. ROIs (yellow boxes) are shown at the right. Scale bar, 0.2 μm.

  • Fig. 4 Depolymerization of microtubules alters the spatial and temporal properties of Ca2+ sparks.

    (A) Representative traces of the fractional increase in fluorescence (F/F0) as a function of time recorded from a Ca2+ spark site within a Fluo-4AM–loaded smooth muscle cell before and after treatment with nocodazole (10 μM). The average time course of Ca2+ spark F/F0 before and after treatment with nocodazole is shown on the right (n = 5 events per group, n = 3 animals). (B) Summary data showing event half-duration [half-time (t1/2), s], rise time (t1/2, s), and decay time (t1/2, s) of Ca2+ sparks recorded before and after treatment with nocodazole. *P ≤ 0.05 compared to control (n = 9 cells, n = 3 animals). (C) Summary data showing Ca2+ spark amplitude (F/F0) before and after treatment with nocodazole (n = 9 cells, n = 3 animals). (D) Representative pseudocolored confocal images of Ca2+ sparks occurring at the same site before and after treatment with nocodazole, illustrating increased spread. Scale bar, 10 μm. Summary data showing spatial spread of Ca2+ sparks before and after treatment with nocodazole are presented on the right. *P ≤ 0.05 compared to control (n = 6 cells, n = 3 animals).

  • Fig. 5 Depolymerization of microtubules decreases the number of BKα and RyR2 colocalization sites.

    (A) Wide-field image of a freshly isolated arterial myocyte immunolabeled for BKα (n = 12 cells, n = 3 animals). Red box indicates the area where superresolution images were obtained. Scale bar, 10 μm. (B) Superresolution localization map obtained after immunolabeling with anti-BKα (green) and anti-RyR2 (red) antibodies (n = 12 cells, n = 3 animals). Scale bar, 3 μm. ROIs (yellow boxes) are shown in magnified view (I) and (II) below. Scale bars, 0.2 μm. (C) Cluster size distribution histograms of RyR2 and BKα (n = 11,005 or n = 11,940 particles for RyR2 and BKα, respectively; 12 cells, n = 3 animals). (D) Summary of object-based analysis to determine the number of BKα protein cluster centroids per cell that overlay with RyR2 protein clusters in control cells, cells in which a random BKα distribution has been simulated, and cells treated with nocodazole (10 μM). *P ≤ 0.05 compared to control (n = 12 cells per group, 3 animals). Coloc., colocalization.

  • Fig. 6 Microtubule-dependent peripheral coupling supports endogenous BK channel activity and regulation of cerebral artery tone by intraluminal pressure.

    (A) Representative perforated voltage-clamp (VH = −30 mV) recording and summary data demonstrating the effects of nocodazole (10 μM) on STOC frequency (n = 5 cells, n = 3 animals; *P ≤ 0.05 compared to control). (B) Representative recordings and summary data demonstrating the effects of nocodazole (10 μM) on single BK channel activity. BK channel activity in inside-out membrane patches was evoked by 3 μM free Ca2+ in the bath solution and was recorded at +40 mV. Nocodazole had no effect on single-channel open probability (NPo) (n = 5 cells, n = 3 animals). (C) Representative recordings of pressure-induced constriction of control- and nocodazole-treated cerebral resistance arteries. Traces show inner luminal diameter at intraluminal pressures between 5 and 100 mmHg for arteries superfused with a Ca2+-containing (black or red traces) or Ca2+-free (blue trace) bathing solution. (D) Summary data showing the effects of nocodazole on myogenic tone as a function of intraluminal pressure (n = 5 arteries, n = 3 animals; *P ≤ 0.05 compared to control). (E) Summary of constriction of control- or nocodazole-treated arteries in response to increased (60 mM) extracellular [K+] (n = 4 arteries, n = 3 animals). (F) Summary concentration-response curves demonstrating that nocodazole treatment does not alter sensitivity to the vasoconstricting thromboxane receptor agonist U46619 (n = 4 arteries, n = 3 animals).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/497/eaan2694/DC1

    Fig. S1. Method used to estimate SR-PM separation distance.

    Fig. S2. Z-stack analysis of interactions between the SR and microtubule arches.

    Fig. S3. Microtubules underlie the peripheral SR.

    Fig. S4. Overlap of RyR2 channel clusters with tubulin.

    Fig. S5. Nocodazole treatment has no effect on total SR Ca2+ store load or Ca2+ spark frequency.

    Fig. S6. BKα and RyR2 nearest neighbor analysis.

    Fig. S7. Actin disruption does not alter STOC frequency.

    Fig. S8. Inhibition of BK channels increases myogenic tone.

    Fig. S9. Effects of nocodazole on passive luminal diameter.

    Movie S1. Microtubule depolymerization increases the separation distance between the SR and the plasma membrane.

    Movie S2. Microtubule structure in freshly isolated smooth muscle cells.

    Movie S3. SR structure in freshly isolated smooth muscle cells.

  • Supplementary Materials for:

    Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility

    Harry A. T. Pritchard, Albert L. Gonzales, Paulo W. Pires, Bernard T. Drumm, Eun A. Ko, Kenton M. Sanders, Grant W. Hennig, Scott Earley*

    *Corresponding author. Email: searley{at}med.unr.edu

    This PDF file includes:

    • Fig. S1. Method used to estimate SR-PM separation distance.
    • Fig. S2. Z-stack analysis of interactions between the SR and microtubule arches.
    • Fig. S3. Microtubules underlie the peripheral SR.
    • Fig. S4. Overlap of RyR2 channel clusters with tubulin.
    • Fig. S5. Nocodazole treatment has no effect on total SR Ca2+ store load or Ca2+ spark frequency.
    • Fig. S6. BKα and RyR2 nearest neighbor analysis.
    • Fig. S7. Actin disruption does not alter STOC frequency.
    • Fig. S8. Inhibition of BK channels increases myogenic tone.
    • Fig. S9. Effects of nocodazole on passive luminal diameter.
    • Legends for movies S1 to S3

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 720 KB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Microtubule depolymerization increases the separation distance between the SR and the plasma membrane.
    • Movie S2 (.mov format). Microtubule structure in freshly isolated smooth muscle cells.
    • Movie S3 (.mov format). SR structure in freshly isolated smooth muscle cells.

    Citation: H. A. T. Pritchard, A. L. Gonzales, P. W. Pires, B. T. Drumm, E. A. Ko, K. M. Sanders, G. W. Hennig, S. Earley, Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility. Sci. Signal. 10, eaan2694 (2017).

    © 2017 American Association for the Advancement of Science

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