Helper and cytotoxic T cells accomplish focused secretion through the movement of vesicles toward the microtubule organizing center (MTOC) and translocation of the MTOC to the target contact site. In this study, using Jurkat cells and OT-I TCR transgenic primary murine CTLs, we show that the dynein-binding proteins nuclear distribution E homolog 1 (NDE1) and dynactin (as represented by p150Glued) form mutually exclusive complexes with dynein, exhibit nonoverlapping distributions in target-stimulated cells, and mediate different transport events. When Jurkat cells expressing a dominant negative form of NDE1 (NDE1–enhanced GFP fusion) were activated by Staphylococcus enterotoxin E–coated Raji cells, NDE1 and dynein failed to accumulate at the immunological synapse (IS) and MTOC translocation was inhibited. Knockdown of NDE1 in Jurkat cells or primary mouse CTLs also inhibited MTOC translocation and CTL-mediated killing. In contrast to NDE1, knockdown of p150Glued, which depleted the alternative dynein/dynactin complex, resulted in impaired accumulation of CTLA4 and granzyme B–containing intracellular vesicles at the IS, whereas MTOC translocation was not affected. Depletion of p150Glued in CTLs also inhibited CTL-mediated lysis. We conclude that the NDE1/Lissencephaly 1 and dynactin complexes separately mediate two key components of T cell–focused secretion, namely translocation of the MTOC and lytic granules to the IS, respectively.
Quantitative PCR (qPCR) was used to investigate whether largemouth bass virus (LMBV) can exist within biofilms. Suspended LMBV was partitioned into either laboratory-grown Pseudomonas fluorescens biofilms or pond-grown, mixed-population biofilms. Biofilm-entrapped LMBV retained infectivity when tested on epithelioma papillosum cyprini tissue culture cells. The LMBV associated with P. fluorescens biofilms were resistant to disinfection by sodium hypochlorite and an iodine-based compound (betadine) but were susceptible to ethanol. Largemouth bass virus was not detected in biofilms or water from ponds that had previously contained LMBV-positive fish, suggesting either that the viral concentrations were below the detection limit of qPCR or that the fish represented the main LMBV reservoir. This study illustrates the potential for LMBV to associate with bacterial biofilms and thereby gain protection from some chemical disinfectants.