Growth, lifetime, directional movement and myosin‑dependent motility of mutant keratin granules in cultured cells
Lehmann SM, Leube RE, Windoffer R
Intermediate filament polypeptides (IFPs) are prominent components of cytoplasmic aggregates, which are pathognomonic for multiple diseases. Recent observations in cultured cells suggest that they are dynamic and subject to regulated turnover. The emerging concept is that multiple factors contribute to motility and turnover of IFP-containing aggregates. To understand their relative contribution, quantitative tools are needed. The current study addresses this need using epithelial cells producing mutant keratin IFPs that have been identified as the cause of the hereditary blister-forming skin disease epidermolysis bullosa simplex. Digital image analysis of individual granules allowed mapping of their complete life cycle, with information on multiple characteristics at any given time-point. The deduced signet features revealed rapid granule fusion and directed transport from the periphery towards the cell centre, and a limited, ~ 30 min lifetime with a slow, continuous growth phase followed by fast disassembly. As paradigmatic proof-of-principle, we demonstrate that inhibition of myosin II selectively reduces granule movement, linking keratin granule motility to retrograde cortical acto-myosin flow. The newly developed methods and established parameters will help in the characterization of known and the identification of novel regulators of IFP-containing aggregates.
Movie 1:
Time-lapse fluorescence recording of EYFP-K14R125C in an MCF-7 cell. Shown is the inverse projection view of 5 focal planes, covering most of the keratin granule signal. Recording frequency: 3 frames/min. Corresponding Fig. 2A shows the still image recorded at time point 0 min.
.gif)
Movie 2:
(A) Image analyses of the time-lapse fluorescence recording of EYFP-K14R125C in the MCF-7 cell depicted in Movie 1. The four quadrants illustrate different stages of image documentation and analysis as described in corresponding Fig. 2B.
(B) The segment is a magnification of (A) (see boxed area in Fig. 2B) and shows the original recordings, the derived tracks of detected granules (magenta circles), the number of granule merging events, and the speed of keratin granules along individual tracks for each time point. Further information is provided in the legend to corresponding Fig. 2C.
.gif){kind=small}
Movie 3:
Image analyses of time-lapse fluorescence recording of EYFP-K14R125C in an MCF-7 cell (recording frequency: 3 frames/min). The track of a single, non-fusing granule is highlighted in green (see also corresponding Fig. 2D). Magenta circles highlight granules from other tracks.
.gif)
.gif)
Movie 5:
Analysis of changing fluorescence (brightness) in a single granule that is tracked in a time-lapse fluorescence recording of EYFP-K14R125C in an MCF-7 cell (corresponding Movie 3). The fluorescence intensity was measured in a single, non-fusing granule moving along the track highlighted in magenta at top (see also corresponding Fig. 4C).
.gif)
Movie 6:
(A) Analysis of changing local track speed, (B) analysis of changing local advance speed, and (C) analysis of changing local advance speed to local track speed ratios of a single granule that is monitored in a time-lapse fluorescence recording of EYFP-K14R125C in an MCF-7 cell (corresponding Movie 3). The values were determined for a single, non-fusing granule moving along the track highlighted in magenta at top (see also corresponding Fig. 5M-0).
.gif)
Movie 7:
Fluorescence recording of EYFP-K14R125C in an MCF-7 cell. Shown are the inverse projection views of 6 focal planes with a total height of 3.44 µm. The cell was incubated in normal medium for 10 min and medium was subsequently replaced with medium containing 20 µM para-nitroblebbistatin until 26 min 20 s (see also corresponding Fig. 7A,B).
.gif)
.gif)
Movie 9:
Fluorescence recording of EYFP-K14R125C mCherry-non-muscle myosin IIB in an MCF-7 cell. Projection view of 4 focal planes with a total height of 1.5 µm. Recording frequency: 3 frames/min. The cell was incubated in normal medium for 7 min and subsequently medium was replaced with medium containing 20 µM para-nitroblebbistatin and imaged for another 25 min 40 s. The keratin track speed was reduced by 24.6% in the treated cell (see also corresponding Fig. 8A,B).
.gif)
Movie 10:
Fluorescence recording of EYFP-K14R125C and LifeAct-RFP in an MCF-7 cell. Projection view of 4 focal planes with a total height of 1.5 µm. Recording frequency: 3 frames/min. The cell was incubated in normal medium for 7 min and subsequently medium was replaced with medium containing 20 µM para-nitroblebbistatin and imaged for another 25 min 40 s. Keratin granule track speed was reduced by 27.8% in the treated cell (see also corresponding Fig. 8C,D).