Actin structures visualized via ß-actin, α-actinin, & actinomyosin

ß-actin

α-actinin

Myosin IIB

Figure. Movies of ß-actin, α-actinin & actinomyosin in various actin structures. Top row movies: Z-stacks of live hiPS cells expressing mEGFP-tagged ß-actin (left), α-actinin (center) and non-muscle myosin heavy chain IIB (right) imaged on a spinning-disk confocal microscope. Images start from the bottom of the cells and end at the top. 

Figure 2. Images of the bottom and top of each structure for easier comparison. Representative images of mEGFP tagged ß-actin (left), α-actinin (center) and non-muscle myosin heavy chain IIB (right) from the bottom (top row) and tops (bottom row) of cells. These images are single slices taken from the z-stacks above. 

Observations
Actin - ß-actin

• ß-actin comprises cytoskeletal filaments that drive protrusion and provide structural support and generate cellular force.
• At the bottom of the cells, ß-actin is both in prominent filaments often associated with cell substrate adhesions and at the periphery of cell protrusions (lamellipodia).
• Near the very top of the cells, ß-actin forms an apical actin band at cell-cell contacts, a feature common in epithelial cells. Small actin puncta of actin are visible along the top surface of cells.
  
• ß-actin is also distributed diffusely throughout the cytoplasm and in brighter foci near cell cell contacts in the center of cells.

Actin bundles - α-actinin

• α-actinin crosslinks actin filaments, forming actin bundles.
• α-actinin localization is almost identical to ß-actin. The main difference is that α-actinin localizes less strongly to the lamellipodia at the protruding edges at the bottom of the cells, where there are fewer bundles.
• Like ß-actin, α-actinin is particularly prominent in the actin-containing band near the top of the cell and in the filamentous structures at the bottom of the cell, along the substratum. 

Actomyosin bundles - Myosin IIB

• Myosin IIB is a contractile motor protein that binds and crosslinks actin filaments.
• Myosin IIB localization is similar to that of actin and α-actinin, residing in prominent filaments primarily at the bottom and top of cells.
• At the bottom of cells it is primarily localized to the contractile stress fibers but not in the protruding regions of the cells.
• Myosin IIB also localizes to the apical actin band found in epithelial cells. However, it is less obviously localized to the actin structures in the center of cells and does not form small puncta at the top surface.

In summary, α-actinin localizes to a subset of actin (e.g. the actin bundles) and myosin IIB to a yet smaller subset (actomyosin bundles) as expected. The general localization pattern of actin containing structures is consistent with an apical-basal epithelial polarity in these cells.