LS-2-O-1870 Interplay between endoplasmic reticulum sheets and dynamic actin filament arrays revealed by multi-scale microscopy
The endoplasmic reticulum (ER) comprises an elaborate 3D network of diverse structural subdomains where functions are distributed according to their specific requirements. The tubule-associated ER functions include interactions with several other cell organelles 1,2,3. Based on the distribution of ER sheet-bound ribosomes4 and the direct correlation between RER abundance and secretion activity of the cell5, protein synthesis, folding and quality control of proteins are functions assigned especially for ER sheets. Proper ER operation requires an intricate balance within and in-between dynamics, morphology, and functions, but how these processes are coupled in cells has remained unclear.
Using multi-scale microscopy comprising of live cell imaging, thin section TEM and two 3D-EM methods, electron tomography and serial block face imaging, we characterized the interplay between ER and the actin cytoskeleton in mammalian cells6. First, we showed that an array of dynamic actin filaments localize in the polygons defined by the surrounding ER structures. Depolymerization of these actin filament arrays led to increased sheet fluctuation and transformations resulting in small and less abundant sheet remnants and a defective ER network distribution. We propose that these filaments have a role in maintaining the sheet-tubule balance and sheet structure by regulating ER sheet remodeling events. Second, we performed a screen where over 200 known functional actin-binding proteins were depleted one-by-one and analyzed by LM to identify those that would have a role on ER morphology and/or dynamics. Based on the screen, we identified the unconventional motor protein myosin 1c (myo1c) localizing to the ER associated actin filament arrays, and revealed a novel role for myo1c in regulating these actin structures. Myo1c depletion and dominant-negative expression of mutated form with abolished actin-binding domain led to loss of the actin filament arrays and to subsequent loss of ER sheets similarly as observed after actin depolymerization using drugs. We propose that ER -associated actin filaments have a role in ER sheet persistence regulation, and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network. In addition to tubular ER associations with microtubules, interactions with the actin cytoskeleton are essential to create and maintain the ER sheet persistence and the characteristic architecture of the ER network in mammalian cells.
1Friedman and Voeltz, Trends Cell Biol. 21 (2011), 709-
2Ylä-Anttila et al., Autophagy 5 (2009),1180-
3Kornmann, Curr.Opin.Cell Biol. 25 (2013), 443-
4Puhka et al., Mol.Biol.Cell 23 (2012), 2424-
5Wiest et al., J.Cell Biol. 110 (1990), 1501-
6Joensuu et al., Mol.Biol.Cell (2014), Epub ahead of print
We thank Mervi Lindman and Antti Salminen for technical assistance. This work was supported by the Academy of Finland (project 131650 to E.J.) and Biocenter Finland. M.J. is a student of the Integrative Life Science doctoral program, University of Helsinki.
Fig. 1: Microtubules and actin filaments can be found in close proximity to ER. High-pressure-frozen, freeze-substituted Huh-7 cells were subjected to electron tomography. From the resulting tomogram, microtubules (blue), actin filaments (red), and ER(yellow) comprising tubules and highly fenestrated sheets were modelled. |
Fig. 2: Time-lapse confocal frames (A) show that the relocation and disappearance of actin arrays (magenta) and ER sheet (green) dynamics are interdependent. ER models (yellow) from control (B) and myo1c-depleted cell (C) reveal consumption of abundant, large and fenestrated sheets (B) into unevenly distributed network of tubules and sheet remnants (C). |