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. 2010 Apr 19.

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 180:887–8951) 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 (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, 5773-83 (2005)

Video 1: Inhibition of the fMLP-induced chemotaxis response in cholesterol-depleted cells. Control cell (left) or MßCD- 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 MßCD (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.