| A critical step in the inflammatory response is transendothelial migration (TEM), the step in which leukocytes squeeze between tightly apposed endothelial cells (EC) to exit the bloodstream and enter inflamed tissue. Our lab has discovered a membrane compartment in EC that regulates TEM: The lateral border recycling compartment (LBRC). The LBRC contains a subset of membrane proteins, such as PECAM, CD99, and JAM-A, which are required for TEM. During TEM, membrane from the LBRC is directed to the region of the EC where the leukocyte is actively transmigrating. This targeted recycling (TR) facilitates TEM by delivering LBRC membrane surface area and signaling molecules to surround the migrating leukocyte. Inhibiting protein interactions required for targeted recycling of the LBRC blocks leukocyte TEM. In addition, inducing microtubule depolymerization in EC blocks TR and consequently, leukocyte TEM as well. Taking it a step further, targeted recycling of the LBRC not only requires functional microtubules, but we have previously shown a requirement for kinesin molecular motors as well. Microinjection of EC with an antibody against the conserved motor domain of kinesin inhibited TR and TEM to a similar magnitude as blocking PECAM homophilic interactions between the leukocyte and EC or depolymerizing microtubules.;We sought to identify the specific kinesin and potential kinesin light chain(s) (and isoform variant(s)) that mediate targeted recycling. Preliminary experiments suggested kinesin-1 as a likely candidate. Knocking down kinesin-1 in HUVEC significantly decreased targeted recycling of LBRC and leukocyte TEM. Microinjection of HUVEC with a kinesin-1 monoclonal antibody significantly blocked TEM and TR. Moreover, knocking down kinesin light chain 1 (KLC1), isoform variant 1 (KLC1C), significantly decreased TEM and TR, whereas knocking down other isoforms of KLC1 had no effect. Our data suggest KLC1C as the adaptor between kinesin-1 and the LBRC during TEM and targeted recycling. This project elucidates the role of kinesin in the regulation of TEM of leukocytes, which is critical for the process of inflammation, and it gives insight into identifying novel potential targets for anti-inflammatory therapy. |
