Poster Sessions

CB -43

Andrius Masedunskas

A. Masedunskas, R. Weigert

Intravital two-photon microscopy for studying the uptake and trafficking of fluorescently conjugated molecules in live rodents

Endocytosis and membrane trafficking have been studied mainly in two-dimensional cell cultures. Although these systems provide a valuable tool to investigate molecular machineries, they do not reproduce the characteristics of the cells in their native tissues. Non-linear imaging techniques such as two-photon microscopy have been increasingly used to visualize tissues in vivo because of reduced photodamage, increased imaging depth and the capacity to detect broad spectrum of fluorescent probes as well as endogenous molecules that provide information on the tissue organization. Here we describe an experimental system based on intravital two-photon microscopy for studying endocytosis in live animals. The rodent submandibular salivary glands were chosen as model organs since they can be exposed easily and imaged without compromising their function. Furthermore, the submandibular glands are amenable to pharmacological and genetic manipulations enabling the study of membrane trafficking at the molecular level. We show that systemically injected molecules such as fluorescently conjugated dextran and bovine serum albumin diffuse out of the vasculature and are rapidly internalized by the supporting cells within the parenchyma of the glands. The cells that internalized fluorescent dextran were labeled by antibody directed against vimentin suggesting their fibroblastic nature. As revealed by time-lapse imaging, the early endosomes undergo homotypic fusion in the periphery of the cells, and within 30-40 minutes reach the late endosomal/lysosomal compartments in the perinuclear area. Notably, heterotypic endosomal-lysosomal fusion was not observed, instead, material from the lysosomes appeared to exchange with endosomal cargo before undergoing fusion with the lysosomal structures. Last, we show that injection of Latrunculin A or Cytochalasin D into the salivary gland via the major excretory duct disrupts the filamentous actin cytoskeleton and markedly reduce the levels of fluorescent dextran uptake. In summary, we demonstrate for the first time that sub-cellular organelles can be dynamically imaged in live animal for extended periods of time. Furthermore, this study establishes that a combination of intravital two-photon microscopy as an imaging technique and the submandibular salivary glands as a model organ can be successfully utilized to study the molecular machinery regulating membrane traffic in live animals.