The actin polymerizing and depolymerizing activities of INF2 are both required for efficient targeting of Lck to the plasma membrane. (A) The alignment shows the conservation of Lys¹⁶⁰¹ in the FH2 domain of Bnp1 in the corresponding position of mDia1, mDia2, and human and mouse INF2 and the position of the 3 Leu residues critical for F-actin depolymerization in the DAD sequence of mouse and human INF2. Note that the third Leu residue is not present in the Bnp1 or mDia1-2 formins. The residues mutated to alanine in the K/A, 3L/A, and K/A-3L/A INF2-1 mutants are indicated. (B-C) Jurkat cells coexpressing GFP and shControl or shINF2b from the same plasmid were cotransfected with expression plasmids encoding intact Cherry fusions of full-length INF2-1 or INF2-1 proteins with the indicated mutations for 48 hours. The expression of these proteins is resistant to shINF2b as their mRNA lack the INF2 3′ untranslated region to which shINF2b is targeted. Cells were fixed, permeabilized, and subjected to immunofluorescence analysis with anti-Lck antibodies and secondary antibodies coupled to Alexa-647 (Lck; B). The staining of INF2-1 was pseudocolored in green and that of GFP in gray to help visualization of the distribution of INF2-1. The histogram shows the mean percentage ± SEM of cells with low levels of peripheral Lck (C). (D) Jurkat cells were treated with 0.1% dimethyl sulfoxide, 1μM Lantrunculin A (LatA), 2.5μM Cytochalasin D (CytD), or 0.5μM Jasplakinolide (Jasp) for 1 hour. Cells were then fixed and stained for F-actin, Lck, and α-tubulin using TRITC-phalloidin and the appropriate primary antibodies followed by secondary antibodies coupled to Alexa-647 (Lck) or Alexa-488 (α-tubulin). (E) The histogram shows the percentage ± SEM of cells with low levels of peripheral Lck. (F) Jurkat cells expressing Lck-GFP for 24 hours were treated with Lantrunculin (LatA) 30 seconds after start, being subjected to time-lapse videomicroscopy. Numbers indicate time in seconds. (G) Jurkat cells expressing GFP fusions of MAL, Lck, or p75 were incubated for the indicated times in the presence of 100μM nocodazole and analyzed under a confocal microscope. The histogram shows the percentage of cells ± SEM with low levels of peripheral MAL, Lck, or p75. The distribution of p75 is presented in gray. The mean ± SEM of 3 independent experiments is represented in panels C,E,G. Staining intensity in was measured in > 50 cells per experiment; 3 independent experiments were performed (*P < .05; **P < .01). Scale bars indicate 5 μm.