Videos

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Virulent Burkholderia species mimic host actin polymerases to drive actin-based motility nucleators
Benanti EL, Nguyen CM, Welch MD.
Cell 161: 348-360 (2015)

Video 1: BmBimA processively binds and elongates filament barbed ends. TIRF microscopy of Alexa Fluor 488-labeled BmBimA (10 nM) elongation of rhodamine-labeled actin filaments. Rhodamine-actin is magenta, and BmBimA is green. Scale bar, 3 μm.

Video 2: BpBimA processively binds and elongates filament barbed ends. TIRF microscopy of Alexa Fluor 488-labeled BpBimA (100 pM) elongation of rhodamine-labeled actin filaments. Rhodamine-actin is magenta, and BpBimA is green. Scale bar, 3 μm.

Video 3: BmBimA elongates two filaments simultaneously. TIRF microscopy of Alexa Fluor 488-labeled BmBimA (10 nM) binding and elongating two actin filaments. Rhodamine-actin is magenta, and BmBimA is green. Scale bars, 3 μm.

Video 4: BmBimA elongates three filaments simultaneously. TIRF microscopy of Alexa Fluor 488-labeled BmBimA (10 nM) binding and elongating three actin filaments. Rhodamine-actin is magenta, and BmBimA is green. Scale bars, 3 μm.

Video 5: BpBimA elongates two filaments simultaneously. TIRF microscopy of Alexa Fluor 488-labeled BpBimA (100 pM) binding and elongating two actin filaments. Rhodamine-actin is magenta, and BpBimA is green. Scale bars, 3 μm.

Video 6: BpBimA elongates three filaments simultaneously. TIRF microscopy of Alexa Fluor 488-labeled BpBimA (100 pM) binding and elongating three actin filaments. The initial two-filament bundle and subsequent three-filament bundle are indicated by arrowheads. Rhodamine-actin is magenta, and BpBimA is green. Scale bars, 3 μm.

Video 7:BimA orthologs mediate distinct modes of actin-based motility in B. thailandensis. From left to right, BtBimA-, BpBimA-, or BmBimA-expressing B. thailandensis infections of Cos7 Lifeact-EGFP cells. Bacteria also constitutively express RFP and are shown in magenta. Lifeact-EGFP is shown in green. Scale bars, 10 μm.


Rickettsia actin-based motility occurs in distinct phases mediated by different actin nucleators
Reed SC, Lamason RL, Risca VI, Abernathy E, Welch MD.
Curr Biol 24: 98-103 (2014)

Video 1: Live HMEC-1 cells transfected with EGFP-Lifeact (green) and infected for 20 min with R. parkeri expressing mCherry (red). Frames were imaged every 5 s; movie plays at 10 frames/s (50x actual speed). Time stamp is in min:s.

Video 2: Live HMEC-1 cells transfected with EGFP-Lifeact (green) and infected for 48 hr with R. parkeri expressing mCherry (red). Frames were imaged every 5 s; movie plays at 10 frames/s (50x actual speed). Time stamp is in min:s.

Video 3: Live HMEC-1 cells stably expressing mCherry-Lifeact (green) and infected for 9 hr with L. monocytogenes 10403S expressing EGFP (red). Frames were imaged every 5 s; movie plays at 10 frames/s (50x actual speed). Time stamp is in min:s.


Regulation of integrin trafficking, cell adhesion, and cell migration by WASH and the Arp2/3 complex
Duleh SN, Welch MD.
Cytoskeleton 69: 1047-1058 (2012)

Video 1: WASH and F-actin dynamics on subdomains of Rab5-Q79L endosomes. NIH3T3 cells transfected with pGFP-WASH (green), pLifeact-BFP (blue) to mark F-actin, and pDsRed-Rab5-Q79L (red) were imaged on a spinning disc confocal microscope.

Video 2: WASH and F-actin dynamics on subdomains of an enlarged endosome. Magnified from Movie 1, corresponding to the inset of Figure 1A.


Rickettsia parkeri invasion of diverse host cells involves an Arp2/3 complex, WAVE complex and Rho-family GTPase-dependent pathway
Reed SC, Serio AW, Welch MD.
Cell Microbiol 14: 529-545 (2012)

Video 1: Actin recruitment around a group of invading Rickettsia. R. parkeri GFP-3 (red) is shown in a Cos7 cell expressing LifeAct-mCherry (green). Images were captured every 10 s starting approximately 23 min post infection and are displayed at 10 frames per second. Bar, 5 μm.

Video 2: Two internalizing bacteria associated with actin. R. parkeri GFP-3 (red) is shown in a Cos7 cell expressing LifeAct-mCherry (green). Images were captured every 10 s starting approximately 30 min post infection and are displayed at 10 frames per second. Bar, 5 μm.

Video 3: Internalization of a bacterium surrounded by mCherry-PAK-PBD. R. parkeri GFP-3 (red) is shown in a Cos7 cell expressing mCherry-PAK-PBD (green). Images were captured every 10 s starting approximately 11 min post infection and are displayed at 10 frames per second. Bar, 5 μm.

Video 4: Internalization of two individual bacteria associated with mCherry-PAK-PBD. R. parkeri GFP-3 (red) is shown in a Cos7 cell expressing LifeAct-mCherry (green). Images were captured every 10 s starting approximately 30 min post infection and are displayed at 10 frames per second. Bar, 5 μm.


The actin nucleation factor JMY is a negative regulator of neuritogenesis
Firat-Karalar EN, Hsiue PP, Welch MD.
Mol Biol Cell 22: 4563-4574 (2011)

Video 1: Polymerization of rhodamine-actin and actin-based motility of beads coated with JMY-WWWCA in Xenopus laevis extract, imaged by epifluorescence microscopy.


Rickettsia Sca2 is a bacterial formin-like mediator of actin-based motility.
Haglund CM, Choe JE, Skau CT, Kovar DR, Welch MD.
Nat Cell Biol 12: 1057-1063 (2010)

Video 1: Polymerization of actin filaments under control conditions, imaged by TIRF microscopy. A red dot marks the pointed end of a control filament (c), and a red open arrowhead marks the growing barbed end.

Video 2: Polymerization of actin filaments in the presence of GST- Sca2, imaged by TIRF microscopy. Green filled arrowheads mark Sca2-associated filaments (s). A red dot marks the pointed end of a control filament (c), and a red open arrowhead marks the growing barbed end.

Video 3: Polymerization of actin filaments in the presence of GST-mDia2(FH1FH2), imaged by TIRF microscopy. A red dot marks the pointed end of a control filament (c), and a red open arrowhead marks the growing barbed end. A blue dot (m) marks the pointed end of an mDia2-associated filament, and a blue open arrowhead marks the growing barbed end.

Video 4: Polymerization of actin filaments in the presence of GST-mDia2(FH1FH2) and profilin, imaged by TIRF microscopy. A blue dot marks the pointed end of an mDia2-bound filament (m), and a blue open arrowhead marks the growing barbed end. A red dot marks the pointed end of a control filament (c), and a red open arrowhead marks the growing barbed end.

Video 5: Polymerization of actin filaments in the presence of profilin, imaged by TIRF microscopy. A red dot marks the pointed end of a control filament (c), and a red open arrowhead marks the growing barbed end.

Video 6: Polymerization of actin filaments in the presence of profilin and GST-Sca2, imaged by TIRF microscopy. A red dot marks the pointed end of a control filament (c), and a red open arrowhead marks the growing barbed end. A green dot marks the pointed end of a Sca2-bound filament (s), and a green open arrowhead marks the growing barbed end.

Video 7: Polymerization of actin filaments in the presence of immobilized GST-mDia2(FH1FH2) and profilin, imaged by TIRF microscopy. A blue dot marks the pointed end of an mDia2-bound filament (m), and a blue open circle marks the growing barbed end.

Video 8: Polymerization of actin filaments in the presence of immobilized GST-Sca2 and profilin, imaged by TIRF microscopy. A green dot marks the pointed end of a Sca2-bound filament (s), and a green open circle marks the growing barbed end.

Video 9: Polymerization of rhodamine-actin on beads coated with GST-Sca2 in Xenopus laevis extract, imaged by epifluorescence microscopy (top) and phase-contrast microscopy (bottom).


Actin-based motility drives baculovirus transit to the nucleus and cell surface.
Ohkawa T, Volkman LE, Welch MD
J Cell Biol 190: 187-195 (2010)

Video 1: Actin-based motility of AcMNPV 3mC virus (red) is shown in a High Five cell expressing EGFP-actin (green).

Video 2: Comparison of the motility of AcMNPV 3mC WT (red; left) and I358A mutant (red; right) in High Five cells expressing EGFP-actin (green).

Video 3: Actin-based motility of AcMNPV 3mC I358A virus (red) in a High Five cell expressing EGFP-actin (green).

Video 4: Comparison of the movement tracks of AcMNPV WT and I358A mutant.

Video 5: AcMNPV 3mC nucleocapsids (red) are shown colliding with the nucleus in High Five cells expressing EGFP- actin (green). The yellow circle highlights a virus colliding with the nucleus, although other collisions are also apparent.

Video 6: AcMNPV 3mC nucleocapsids (red) are shown colliding with the nucleus in High Five cells expressing EGFP- actin (green).

Video 7: EGFP-actin is shown in High Five cells 5 min after infection. The yellow circle highlights a virus colliding with the nucleus, although other collisions are also apparent. After collisions, virus-associated actin comet tails persist, resembling corkscrews that radiate from the nuclear periphery.

Video 8: AcMNPV 3mC nucleocapsids (red) are shown separating from their actin comets and entering the nucleus in High Five cells expressing EGFP-actin (green). After nucleocapsid entry into the nucleus, actin polymerization ceases.

Video 9: An AcMNPV 3mC I358A nucleocapsid (red) is shown with a virus-associated actin corkscrew structure radiating from the nuclear periphery in High Five cells expressing EGFP-actin (green).

Video 10: AcMNPV 3mC virus (red) is shown moving into surface spikes in a High Five cell expressing EGFP-actin (green). This video is a magnified and cropped view from Video 1.


Defining a core set of actin cytoskeletal proteins critical for actin- based motility of Rickettsia.
Serio AW, Jeng RL, Haglund CM, Reed SC, Welch MD.
Cell Host Microbe 7: 388-398 (2010)

Video 1:Actin labeled with Lifeact-GFP in Rickettsia parkeri infected S2R+ cells not treated with dsRNA (left) or treated with dsRNA targeting capping protein (right).

Video 2: Actin labeled with Lifeact-GFP in Rickettsia parkeri infected S2R+ cells treated with dsRNA targeting cofilin (left) or fimbrin (right).

Video 3:Actin labeled with Lifeact-GFP in Rickettsia parkeri infected S2R+ cells treated with dsRNA targeting profilin, showing a short tail phenotype (left) or short actin spikes (right).

Video 4: Plastin-GFP in COS7 cells infected with Rickettsia parkeri (left) or Listeria monocytogenes (right).

Video 5:Profilin-GFP in COS7 cells infected with Rickettsia parkeri (left) or Listeria monocytogenes (right).

Video 6: GFP-capping protein in COS7 cells infected with Rickettsia parkeri (left) or Listeria monocytogenes (right).

Video 7: GFP-cofilin in COS7 cells infected with Rickettsia parkeri (left) or Listeria monocytogenes (right).

Video 8: Phase contrast images (top) or GFP fluorescence (bottom) in COS7 cells infected with Rickettsia parkeri (left) or Listeria monocytogenes (right).

Video 9: Actin labeled with Lifeact-GFP in a Rickettsia parkeri infected COS7 cell transfected with a control non- specific siRNA.

Video 10: Actin labeled with Lifeact-GFP in a Rickettsia parkeri infected COS7 cell depleted of T-plastin by RNAi.

Video 11: Actin labeled with Lifeact-GFP in a Rickettsia parkeri infected COS7 cell depleted of profilin 1 by RNAi.


An actin-filament-binding interface on the Arp2/3 complex is critical for nucleation and branch stability.
Goley ED, Rammohan A, Znameroski EA, Firat-Karalar EN, Sept D, Welch MD.
Proc Natl Acad Sci U S A 107: 8159-8164 (2010)

Video 1: Three-dimensional view comparing models of the interaction between ARPC2/ARPC4 and F actin. The position of ARPC2/ARPC4 derived from protein-protein docking simulations is depicted in pink and cyan, whereas the position of these subunits suggested by Rouiller et al. (2008, J Cell Biol 180:887-895) is shown in yellow. The actin filament is depicted in white.


WASH and the Arp2/3 complex regulate endosome shape and trafficking.
Duleh SN, Welch MD.
Cytoskeleton 67: 193-206 (2010)

Video 1: WASH (green) is asymmetrically distributed on EEA1-positive endosomes (red) as visualized by deconvolution microscopy and 3D reconstruction.


WHAMM is an Arp2/3 complex activator that binds microtubules and functions in ER to Golgi transport.
Campellone KG, Webb NJ, Znameroski EA, Welch MD.
Cell 134: 148-161 (2008)

Video 1: Dynamics of GFP-WHAMM-associated membranes in a Cos7 cell over a 10 min timecourse.

Video 2: Dynamics of GFP-WHAMM-associated membranes in a Cos7 cell over a 6 min timecourse.

Video 3: Dynamics of GFP-WHAMM-associated membranes in an NIH 3T3 cell in the presence of nocodazole. The drug was added 3 min into a 9 min time series.

Video 4: Dynamics of GFP-WHAMM-associated membranes in an NIH 3T3 cell in the presence of cytochalasin D. The drug was added 3 min into a 9 min time series.

Video 5: Dynamics of GFP-WHAMM-associated membranes in an NIH 3T3 cell in the presence of latrunculin A. The drug was added 3 min into a 9 min time series.

Video 6: Dynamics of GFP-WAMM(W807A)-associated tubules in an NIH 3T3 cell over a 6 min timecourse.


Dynamic nuclear actin assembly by Arp2/3 complex and a baculovirus WASP-like protein.
Goley ED, Ohkawa T, Mancuso J, Woodruff JB, D'Alessio JA, Cande WZ, Volkman LE, Welch MD
Science 314: 464-467 (2006)

Video 1: Actin localization and polymerization in infected TN-368 cells expressing EGFP-actin, viewed from 14:30 to 21:42 hours post infection (hpi). Latrunculin A is added at approximately 20 hpi, causing depolymerization of the discrete nuclear F-actin structures.

Video 2: Actin localization and polymerization in infected TN-368 cells expressing EGFP-actin, viewed from 18:45 to 22:05 hours post infection.

Video 3: Actin localization and polymerization in infected TN-368 cells expressing mCherry-actin, viewed from 5:44 to 22:40 hours post infection.

Video 4: Fluorescence recovery after photobleaching a region of the nucleus in an infected TN-368 cell expressing EGFP-actin. The circle marks the photobleached region.

Video 5: Fluorescence loss in photobleaching of nuclear GFP-actin in a representative infected TN-368 cell that has been treated with latrunculin A.


Plasma membrane organization is essential for balancing competing pseudopod- and uropod-promoting signals during neutrophil polarization and migration.
Bodin S, Welch MD.
Mol Biol Cell 16, 5773-5783 (2005)

Video 1: Inhibition of the fMLP-induced chemotaxis response in cholesterol-depleted cells. Control cell (left) or MβCD-treated cell (right) were visualized using DIC microscopy.

Video 2: Cholesterol-depletion increases the chemoattractant sensitivity of the rear edge. Cell was visualized using DIC microscopy.

Video 3: Cholesterol depletion does not inhibit the fMLP-induced formation of a retracting uropod in pertussis toxin- treated cells. Pertussis-toxin (PTX) treated cell (left) and a cell treated with both PTX and MβCD (right), visualized using DIC microscopy.

Video4, Video 5, Video 6: Cholesterol is required to restrict D3-PI synthesis to the pseudopod. Control cell (Video 4) and cholesterol-depleted cells (Videos 5 and 6) expressing PH-Akt-GFP, left panels DIC microscopy, right panels PH- Akt-GFP fluorescence.

Video 7: Distribution of D3-PIs in a control cell during an abrupt reversion of the fMLP gradient visualized by PH-Akt- GFP fluorescence.

Video 8: Distribution of D3-PIs in a cholesterol- depleted cell during an abrupt reversion of the fMLP-gradient visualized by PH-Akt-GFP fluorescence.

Video 9: Cholesterol depletion induces pseudopod formation in a PI3-K- and Gi- dependent manner. Cells expressing PH- Akt-GFP were exposed to a gradient of MbCD diffusing from a micropipette. Cells respond by emitting a pseudopod facing the pipette tip (top panel), accompanied by a modest membrane relocation of PH-Akt-GFP (indicated by white arrow on frame 100 s). This response is prevented in cells pre-treated by pertussis-toxin (middle panel), or wortmannin (lower panel).


A Rickettsia WASP-like protein activates the Arp2/3 complex and mediates actin-based motility.
Robert L. Jeng, Erin D. Goley, Joseph A. D'Alessio, Oleg Y. Chaga, Tatyana M. Svitkina, Gary G. Borisy, Robert A. Heinzen and Matthew D. Welch.
Cell Microbiol 6: 761-769 (2004)

Video 1 Movie of actin structures assembled by RickA-coated beads in Xenopus egg extract that was supplemented with rhodamine-labelled actin and visualized by fluorescence microscopy.

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