A role for myosin II in mammalian mitochondrial fission

Abstract

Mitochondria are dynamic organelles, undergoing both fission and fusion regularly in interphase cells. Mitochondrial fission is thought to be part of a quality-control mechanism whereby damaged mitochondrial components are segregated from healthy components in an individual mitochondrion, followed by mitochondrial fission and degradation of the damaged daughter mitochondrion. Fission also plays a role in apoptosis. Defects in mitochondrial dynamics can lead to neurodegenerative diseases such as Alzheimer's disease. Mitochondrial fission requires the dynamin GTPase Drp1, which assembles in a ring around the mitochondrion and appears to constrict both outer and inner mitochondrial membranes. However, mechanisms controlling Drp1 assembly on mammalian mitochondria are unclear. Recent results show that actin polymerization, driven by the endoplasmic reticulum-bound formin protein INF2, stimulates Drp1 assembly at fission sites. Here, we show that myosin II also plays a role in fission. Chemical inhibition by blebbistatin or small interfering RNA (siRNA)-mediated suppression of myosin IIA or myosin IIB causes an increase in mitochondrial length in both control cells and cells expressing constitutively active INF2. Active myosin II accumulates in puncta on mitochondria in an actin- and INF2-dependent manner. In addition, myosin II inhibition decreases Drp1 association with mitochondria. Based on these results, we propose a mechanistic model in which INF2-mediated actin polymerization leads to myosin II recruitment and constriction at the fission site, enhancing subsequent Drp1 accumulation and fission.

Figure 1. Blebbistatin treatment increases mitochondria length in U2OS cells

(A) Control U2OS cells (top) or cells expressing GFP-INF2 A149D (bottom) were treated with DMSO (left) or 50 µM blebbistatin (right) for 60 min, then stained with Mitotracker. Asterisks indicate cells expressing INF2 A149D (See Figure S1 for GFP and actin staining). Scale bar, 10 µm. (B) Quantification of mitochondrial length in control or GFP-INF2 A149D expressing cells, treated with DMSO or 50 µM blebbistatin for indicated times. N=103 to 344 mitochondria. Asterisks denote p values of < 0.01 by Student’s T-test. Error bars, SEM. (C) Quantification of mitochondial length in control cells and cells treated with 200 µM CK689 (negative control compound) or CK666 (Arp2/3 complex inhibitor) for 60 min. N=223 to 289 mitochondria. Error bars, SEM.

Figure 2. Depletion of myosin IIA or IIB increases mitochondrial length

(A) Mitotracker staining of U2OS cells expressing indicated siRNAs. Scale bar, 10 µm. (B) U2OS cells expressing control or myosin IIB siRNA were transfected with GFPINF2 A149D (green) and stained with mitotracker (red). Mitochondria are alone also shown separately in the right panels. (C) and (D) Quantification of mitochondrial lengths for experiments depicted in A and B, respectively. Data from three experiments, n = 261 to 368 mitochondria for C, and 329 to 407 mitochondria for D. Error bars, SEM.

Figure 3. Myosin II enriches at mitochondrial constriction sites

(A) U2OS cell transfected with mito-BFP and ER-green, then fixed and stained with anti-P-MRLC (myosin regulatory light chain, phosphoserine 19). Arrowhead shows mitochondrial constriction site. Scale bar, 10 µm. (B) U2OS cells labeled with mitotracker (red), then fixed, stained with anti-PMRLC (green), and imaged by spinning disc confocal microscopy with 5–7 z-sections taken (200 nm z steps). Arrowheads denote constriction sites. Two examples shown. For each, left is maximum intensity image and right is 3D reconstruction. Scale bar, 2 µm. (C) U2OS cell transfected with mito-dsRed and GFP-MIIA, then imaged live by confocal microscopy (single Z-plane). Arrow points to MIIA accumulation at fission site. Scale bar, 2 µm. See also Movie S1. (D) Single confocal slices of “apical” mitochondria, located adjacent to the nucleus at > 1.4 µm from the ventral surface of the cell, avoiding interference from abundant ventral stress fibers. U2OS cells labeled with mitotracker (red), then fixed and stained with anti-P-MRLC (green). Cells either untreated (left), transfected with siRNA oligos against INF2 for 72 hrs (middle), or treated with 0.5 µM Latrunculin B for 60 min (right). Scale bar, 20 µm. See Figure S3 for quantification of mitochondria-associated P-MRLC puncta upon Myosin II and mitochondrial fission these treatments, as well as close-up image of apical mitochondria, P-MRLC, and actin filaments.

Figure 4. Localization of DRP1 to mitochondria is reduced upon myosin II suppression

(A) U2OS cells transfected with control, MIIA or MIIB siRNAs were treated with mitotracker (red), then fixed and immunostained for DRP1 (green). Zoomed regions shown on bottom row. Scale bar, 10 µm. B) Quantification of mitochondrial-associated DRP1 puncta in control and myosin II suppressed cells. Data from two experiments, n= 87 to 110 mitochondria. Error bars, SEM.