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Cell Migration pp 257-277 | Cite as

Imaging the Molecular Machines That Power Cell Migration

  • Anika Steffen
  • Frieda Kage
  • Klemens RottnerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1749)

Abstract

Animal cell migration constitutes a complex process involving a multitude of forces generated and maintained by the actin cytoskeleton. Dynamic changes of the cell surface, for instance to effect cell edge protrusion, are at the core of initiating migratory processes, both in tissue culture models and whole animals. Here we sketch different aspects of imaging representative molecular constituents in such actin-driven processes, which power and regulate the polymerisation of actin filaments into bundles and networks, constituting the building blocks of such protrusions. The examples presented illustrate both the diversity of subcellular distributions of distinct molecular components, according to their function, and the complexity of dynamic changes in protrusion size, shape, and/or orientation in 3D. Considering these dynamics helps mechanistically connecting subcellular distributions of molecular machines driving protrusion and migration with their biochemical function.

Key words

Live cell imaging Actin Migration WAVE complex VASP FMNL Fluorescence imaging 

Notes

Acknowledgments

This work was supported in part by the German Research Foundation (DFG) and by the Helmholtz Centre for Infection Research (HZI, Braunschweig, Germany). We would like to thank Dr. Yu-Li Wang (Pittsburgh, USA) for insightful discussions on fluorescence imaging.

Supplementary material

429754_1_En_19_MOESM1_ESM.mp4 (1.3 mb)
Movie 1 EGFP-VASP accumulates in protruding lamellipodia. Time-lapse epifluorescence and phase-contrast microscopy reveals VASP to be enriched at the lamellipodial front as long as the edge protrudes forward, while it disappears from the focal plane in local phases of upward and rearward ruffling and/or retraction. EGFP-VASP also accumulates in protruding filopodia and focal adhesions. Time is displayed in minutes and seconds (AVI 2021 kb)
429754_1_En_19_MOESM2_ESM.mp4 (883 kb)
Movie 2 Filopodia can emerge from ruffling lamellipodia. Time-lapse fluorescence and phase-contrast microscopy of B16-F1 cell transfected with EGFP-VASP, which is enriched at lamellipodia rims, filopodia tips, and focal adhesions. Upper panel shows an overview of the cell, grey box in phase-contrast movie marks the zoom region that is shown in higher magnification below. Note that when the lamellipodium starts to ruffle, the VASP signal transiently vanishes from the front because of leaving the focal plane, but comes back as soon as the rearward folded lamellipodium returns into the focal plane. At this point, the ruffling edge of the rearward-folded lamellipodium develops and protrudes a filopodium in rearward direction of migration. Simultaneously, a new lamellipodium protrudes from the front, which accumulates EGFP-VASP signal, and the cycle of protrusion and ruffling and/or retraction starts from the beginning. All these activities highlight the complexity of the dynamics of molecules operating in actin filament regulation in cell edge protrusions. Time is shown in minutes and seconds (AVI 1608 kb)
429754_1_En_19_MOESM3_ESM.mp4 (2.1 mb)
Movie 3 FMNL2-EGFP localizes to the tip regions of lamellipodia and filopodia. Time-lapse fluorescence and phase-contrast microscopy of B16-F1 cell migrating on laminin and transiently transfected with FMNL2 tagged with EGFP at its C-terminus (FMNL2-EGFP). Note that formin accumulation is restricted to protruding lamellipodia or filopodia. It is absent from retracting regions of the cell. Time is given in minutes and seconds (AVI 3037 kb)
429754_1_En_19_MOESM4_ESM.mp4 (247 kb)
Movie 4 Full length EGFP-FMNL2 displays cytosolic localization. B16-F1 cell transiently transfected with EGFP-tagged full length FMNL2. Time-lapse epifluorescence imaging reveals a mostly cytosolic localization of the fusion protein due to inhibition of myristoylation by N-terminal EGFP-tagging [13]. Note the stark contrast with C-terminal tagging displayed in Movie 3. Time is given in minutes and seconds (AVI 635 kb)
429754_1_En_19_MOESM5_ESM.mp4 (5 mb)
Movie 5 EGFP-CP localizes to the lamellipodium and endosomes. The movie shows a NIH3T3 cell expressing EGFP-CP. Fluorescence (left) and phase-contrast (right) time-lapse microscopy of the whole cell (top panels) and details of the regions (bottom panels) as indicated at the top. The cell was co-transfected with myc-Rac1-L61, leading to constitutive treadmilling of lamellipodia at the periphery as well as at the ventral side. CP is localized to the lamellipodium, as well as ruffles and endosomes. It is difficult in these conditions to differentiate small, ventral ruffles from endosomes, except that those structures appearing at the periphery are likely ruffles since the fluorescent accumulation of CP is accompanied by fuzzy dark-grey structures in phase contrast, more likely corresponding to ruffles. Endosomes, instead, appear as white, grey or dark grey round vesicular structures (see top right panel). Note that co-expression of appropriate marker proteins would facilitate identifying structures like endosomes and ruffles, or distinguishing them from one another. Note that upon ruffling, CP seems to enrich at the extreme periphery; however, this can be accounted for by the transient, local thickening of the cell periphery, as revealed in corresponding phase-contrast time lapse images, and not in this case by a real restriction to the lamellipodium tip, as shown with other components such as Abi1 (Fig. 5 and Movie 6). Time is given in minutes and seconds (AVI 7317 kb)
429754_1_En_19_MOESM6_ESM.mp4 (4.3 mb)
Movie 6 EGFP-Abi1 exclusively accumulates at the tips of protruding lamellipodia. The movie shows a NIH3T3 cell expressing EGFP-Abi1. Fluorescence (left) and phase-contrast (right) time-lapse microscopy of the whole cell (top panels) and details of the regions as indicated at the top in bottom panels. The cell was co-transfected with myc-Rac1-L61, leading to constitutive treadmilling of lamellipodia at the periphery as well as at the ventral side. Note that EGFP-Abi1 is localized at the extreme tip of the periphery. As shown in the details at the bottom, the lamellipodium is frequently lifting up, which can be appreciated by a fuzzy periphery in phase contrast. The EGFP-Abi1 signal is disappearing correspondingly, due to out of focus localization. Protrusive ventral lamellipodia can be seen in both fluorescence as well as phase-contrast channels. Time is given in minutes and seconds (AVI 6078 kb)
429754_1_En_19_MOESM7_ESM.mp4 (2.2 mb)
Movie 7 Control NIH3T3 cell expressing EGFP. Fluorescence (left) and phase-contrast (right) time-lapse microscopy of the whole cell (top panel) and details of the regions as indicated (bottom panel) are shown. Time is given in minutes and seconds. Note that when the periphery of the cell is ruffling, the cytosolic EGFP signal seems to correspond to a distinct “localization” at the periphery, however, this is just due to the increased thickness of the cell as illustrated also by phase-contrast microscopy. In images using the latter, thicker and/or denser regions of the cell appear darker, whereas very dense material appears as dark spots, and fatty or liquid-containing vesicles such as macropinosomes constitute white structures (AVI 4714 kb)

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.Department of Cell BiologyHelmholtz Centre for Infection ResearchBraunschweigGermany
  2. 2.Division of Molecular Cell Biology, Zoological InstituteTechnische Universität BraunschweigBraunschweigGermany

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