Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Re-evaluating the roles of myosin 18Aα and F-actin in determining Golgi morphology

doi: 10.1002/cm.21364

Figure Lengend Snippet: Endogenous M18Aα does not localize to the Golgi. (a) Wild-type (WT) MEF fixed and stained for F-actin (phalloidin, a1) and M18A (Antibody #1, a2). The merged image in (a3) includes the signal for the nucleus (DAPI, blue). The position of the nucleus is marked with a yellow dashed line in (a1 and a2). White arrowheads mark cortical regions where M18A and actin are enriched. The images are maximum intensity projections of the bottom five 0.236 µm slices from a z-stack acquired on a Nikon A1R confocal microscope (“Basal”). Scale bar, 20 µm. (b) Exactly as in (a) except using Antibody #2. (c) WT MEF fixed and stained for M18A (Antibody #1, c1 and c2) and the Golgi (GM130, c3). The merged image in (c4) includes the signal for the nucleus (DAPI, blue). The position of the nucleus is marked with a yellow dashed line in (c1). White arrowheads in (c2) mark cortical regions where M18A and actin are enriched. (c1) is a maximum intensity projection of the bottom five 0.236 µm slices from the full z-stack (“Basal”). (c2–c4) are maximum intensity projections of all 0.236 µm slices acquired on a Nikon A1R confocal microscope (“Full Z Projection”). Scale bar, 10 µm. (d) Exactly as in (c) except using Antibody #2. The apparent co-localization between M18A and the Golgi is marked with red arrows. (e and f) Exactly as in (c and d) except using a M18A KO MEF. Note that the apparent colocalization between M18A and the Golgi (red arrows) seen with Antibody #2 in (d) persists in cells lacking M18Aα and M18Aβ in (f) [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: White arrowheads in (c2) mark cortical regions where M18A and actin are enriched. (c1) is a maximum intensity projection of the bottom five 0.236 µm slices from the full z-stack (“Basal”). (c2–c4) are maximum intensity projections of all 0.236 µm slices acquired on a Nikon A1R confocal microscope (“Full Z Projection”).

Techniques: Staining, Microscopy

Journal: Cytoskeleton (Hoboken, N.J.)

Article Title: Re-evaluating the roles of myosin 18Aα and F-actin in determining Golgi morphology

doi: 10.1002/cm.21364

Figure Lengend Snippet: Abrogation of M18Aα expression does not alter Golgi morphology, which is highly variable. (a and b) Control HeLa cells expressing non-targeting (NT) shRNA (NT HeLa, a1 and a2) and Hela cells expressing M18A shRNA (M18A KD HeLa, b1 and b2) were fixed and stained with the cis-Golgi marker α-GM130 (green) and the nuclear marker DAPI (blue). The images are maximum intensity projections of wide-field z-stacks acquired on a Deltavision OMX microscope with 0.125 µm steps. These representative images show that the Golgi can exist in both extended and contracted states in both control and M18A KD cells. Scale bar, 5 µm. (c) Golgi size, quantified as a fraction of nuclear perimeter, was measured in the indicated cell types prepared and was imaged as in (a) and (b). The n values are as follows: NT HeLa (47), M18A KD HeLa (55), WT Rat2 (39), M18A KO Rat2 (49), WT MEF (49), and M18A KO MEF (43). (d) NT HeLa cell expressing mCherry-H2B to mark the nucleus (red) and Mann II-mEmerald to mark the Golgi (green) and was imaged using a Nikon A1R microscope. Shown are representative examples of the variations in Golgi morphology exhibited by this cell over time (A/P values are shown in the upper right corner). Scale bar, 10 µm. (e) A/P values for 15 randomly chosen NT HeLa cells (grey) imaged as described in (d) every 10 min over 12 hr. (f) Exactly as in (e) but with M18A KD HeLa cells (red). (g) Means and standard deviations for all the A/P values obtained over 12 hr for NT HeLa (grey) and M18A KD HeLa (red). (H) Means and standard deviations for the summed changes in A/P values over 12 hr for NT HeLa (grey) and M18A KD HeLa (red). (i) Means and standard deviations for the rates of change in A/P values per 10-min interval going from either collapsed to extended and or from extended to collapsed for NT HeLa (grey) and M18A KD HeLa (red). Where appropriate, p values are indicated (n.s. indicates a lack of significance) [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: White arrowheads in (c2) mark cortical regions where M18A and actin are enriched. (c1) is a maximum intensity projection of the bottom five 0.236 µm slices from the full z-stack (“Basal”). (c2–c4) are maximum intensity projections of all 0.236 µm slices acquired on a Nikon A1R confocal microscope (“Full Z Projection”).

Techniques: Expressing, shRNA, Staining, Marker, Microscopy