Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections

Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility

Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections

Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility

Supplemental Figure 3. Palladin Recruitment at L. monocytogenes Requires ActA.m

HeLa cells were infected with L. monocytogenes strain ΔactA for 6 hours, fixed and stained with a mouse monoclonal palladin targeting antibody (green), DAPI (blue) to visualize DNA and Alexa594-phalloidin (red) to visualize actin. Palladin is absent from the surface of intracellular bacteria (boxed regions). Insets (enlargement of boxed regions) points to an individual bacterium (open arrowhead). Scale bar, 10 µm and 2 µm (insets)

Figure S4. Actin-Rich Cytoplasmic Clumps Are Generated in PalladinFPAA-Expressing Cells.

<p>HeLa cells were transfected with various GFP-tagged palladin DNA constructs (indicated on left), fixed and stained with DAPI (blue) to visualize DNA and Alexa594-phalloidin to visualize actin and simultaneously observe for general cytoskeletal alterations. </p> <p>(A) Palladin displays robust punctate staining at actin stress fibers in GFP-palladin transfected cells. In GFP-palladinFPAA cells, actin stress fibers are generally absent from within the central body of the cell and abnormal actin-rich cytoplasmic clumps (open arrowheads) and bar-like structures (solid arrowheads) are formed. Palladin retains some punctate staining at residual stress fibers within the cell. The actin cytoskeleton is disorganized and palladin is redistributed to the nucleus in GFP-palladinK15/18/51A transfected cells. Scale bar, 5 µm (also applies to B).</p> <p>(B) Endogenous VASP (red) is redistributed to actin-rich cytoplasmic clumps and bar-like structures formed in GFP-palladinFPAA transfected cells.</p

Figure S3. Palladin Recruitment at L. monocytogenes Requires ActA.

<p>HeLa cells were infected with <i>L. monocytogenes </i>strain Δ<i>actA </i>for 6 hours, fixed and stained with a mouse monoclonal palladin targeting antibody (green), DAPI (blue) to visualize DNA and Alexa594-phalloidin (red) to visualize actin. Palladin is absent from the surface of intracellular bacteria (boxed regions). Insets (enlargement of boxed regions) points to an individual bacterium (open arrowhead). Scale bar, 10 µm and 2 µm (insets).</p

Figure S6. PalladinFPAA fails to nucleate actin clouds and comet tails in the absence of the Arp2/3 complex.

<p><i>L .monocytogenes</i> bacteria were incubated in a motility medium (with rhodamine-labelled G-actin in red) reaction containing palladinFPAA in place of the Arp2/3 complex. PalladinFPAA polymerizes F-actin bundles (top) and disorganized F-actin clumps (bottom). <i>L .monocytogenes</i> bacteria (open arrows) do not polymerize F-actin at their surface. Scale bar, 10 µm.</p

Supplemental Figure 4. Actin-Rich Cytoplasmic Clumps Are Generated in PalladinFPAA-Expressing Cells.

<p>HeLa cells were transfected with various GFP-tagged palladin DNA constructs (indicated on left), fixed and stained with DAPI (blue) to visualize DNA and Alexa594-phalloidin to visualize actin and simultaneously observe for general cytoskeletal alterations. </p> <p>(A) Palladin displays robust punctate staining at actin stress fibers in GFP-palladin transfected cells. In GFP-palladinFPAA cells, actin stress fibers are generally absent from within the central body of the cell and abnormal actin-rich cytoplasmic clumps (open arrowheads) and bar-like structures (solid arrowheads) are formed. Palladin retains some punctate staining at residual stress fibers within the cell. The actin cytoskeleton is disorganized and palladin is redistributed to the nucleus in GFP-palladinK15/18/51A transfected cells. Scale bar, 5 µm (also applies to B).</p> <p>(B) Endogenous VASP (red) is redistributed to actin-rich cytoplasmic clumps and bar-like structures formed in GFP-palladinFPAA transfected cells.</p

Figure S5. Tethered palladin-generated comet tail in the absence of the Arp2/3 complex.

<p><i>L .monocytogenes</i> bacteria were incubated in a motility medium (with rhodamine-labelled G-actin in red) reaction containing full length palladin in place of the Arp2/3 complex. An F-actin cloud surrounding the bacterial cell (open arrow) is trailed by a palladin-generated comet tail (solid arrows) that appears to be tethered to a clump of F-actin (star) that resembles those formed in cells expressing GFP-palladinFPAA.</p

Figure S2. L. monocytogenes Invasion and Listeriopods Are Unaffected by Palladin Depletion.

<p>HeLa cells were treated with non-targeting control (Ctrl) or palladin-targeted (KD) siRNA sequences and infected with wild type <i>L. monocytogenes</i>. </p> <p>(A) HeLa cells were fixed following 3 hour infections and stained with a mouse monoclonal palladin targeting antibody (green), DAPI (blue) to visualize DNA and Alexa594-phalloidin (red) to visualize actin. Bacterial internalization efficiency was analyzed by counting and comparing the number of total internalized bacteria in Ctrl and KD (no detectable palladin observed by immunofluorescence microscopy) cells. In 3 independent experiments, 951 bacteria were counted in a total of 10 Ctrl cells and 822 bacteria were counted in a total of 10 KD cells; for each experiment all counts were normalized to the Ctrl counts. Data depicts percentage of bacteria invasion compared to Ctrl cells.</p> <p>(B) Palladin-depleted cells were fixed following 6 hour infections and stained with a mouse monoclonal palladin targeting antibody (green), DAPI (blue) to visualize DNA and Alexa594-phalloidin (red) to visualize actin. Listeriopod formation (arrow) is unaffected by palladin depletion. Insets depict enlargement of region indicated by arrow and shows listeriopod of interest (open arrowhead). Scale bars, 5 µm and 1 µm (insets).</p

Supplemental Figure 5. Tethered palladin-generated comet tail in the absence of the Arp2/3 complex.

<p><i>L .monocytogenes</i> bacteria were incubated in a motility medium (with rhodamine-labelled G-actin in red) reaction containing full length palladin in place of the Arp2/3 complex. An F-actin cloud surrounding the bacterial cell (open arrow) is trailed by a palladin-generated comet tail (solid arrows) that appears to be tethered to a clump of F-actin (star) that resembles those formed in cells expressing GFP-palladinFPAA.</p