Exosomes are microvesicles of endosomal origin that are secreted, and their contents (proteins, lipids, DNA, or microRNAs) can alter the physiological states of recipient cells. We demonstrated that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein normally localized in the cytoplasm and nucleus, was secreted in exosomes. Secreted PTEN was internalized by recipient cells with resultant functional activity, which resulted in reduced phosphorylation of the serine and threonine kinase Akt and reduced cellular proliferation. PTEN secretion in exosomes required Ndfip1, an adaptor protein for members of the Nedd4 family of E3 ubiquitin ligases. Without Ndfip1, neither Nedd4-1 nor Nedd4-2 promoted the recruitment of PTEN into exosomes. In addition, lysine 13 within PTEN, which is required for its ubiquitination by Nedd4-1, was required for exosomal transport of PTEN. These results implicate Ndfip1 as a molecular regulator of the exosomal export of PTEN, with consequences for non-cell-autonomous PTEN activity. Thus, we suggest that the ability of PTEN to exert phosphatase activity beyond the cell in which it is produced has implications for PTEN function during development, health, and disease.

The nervous system has a modular architecture with neurons of the same type commonly organized in nonrandom arrays or mosaics. Modularity is essential to parallel processing of sensory information and has provided a key element for brain evolution, but we still know very little of the way neuronal mosaics form during development. Here we have identified the immature elements of two retinal mosaics, the choline acetyltransferase (ChAT) amacrine cells, by their early expression of the homeodomain protein Islet-1, and we show that spatial ordering is an intrinsic property of the two Islet-1 mosaics, dynamically maintained while new elements are inserted into the mosaics. Migrating Islet-1 cells do not show this spatial ordering, indicating that they must move tangentially as they enter the mosaic, under the action of local mechanisms. Clonal territory analysis in X-inactivation transgenic mice confirms the lateral displacement of ChAT amacrine cells away from their clonal columns of origin, and mathematical models show how short-range cellular interactions can guide the assemblage of these mosaics via a simple biological rule. Key words: retina; LIM proteins; Islet-1; X-inactivation transgenic mouse; ChAT amacrine; tangential migration; Voronoi domainsThe retina is one of the best examples of modular organization in neural circuitry. The five main types of retinal neurons are organized into three cell layers. Photoreceptors occupy the outer nuclear layer, bipolar, horizontal, and amacrine cells the inner nuclear layer (I N L), and ganglion cells and displaced amacrine cells the ganglion cell layer (GCL). Each principal class of retinal neurons can be divided f urther into subtypes, which differ in morphology and connectivity as well as biochemical and physiological properties (for review, see Ramon y C ajal, 1892;Rodieck, 1973;Dowling, 1987;Wässle and Boycott, 1991). Within each layer, neurons of the same type are commonly spaced in an orderly manner, forming planar arrays that uniformly tile the retina. Such arrays are known as neuronal mosaics (Wässle and Riemann, 1978) because they bring to mind the regular arrangement of the tesserae of a mosaic.Although the orderly organization of retinal cells is known to be f undamental to the parallel processing of visual information in the retina, little is known of the way neuronal mosaics form during development. Postmitotic retinal neurons migrate to their final positions from the proliferative neuroepithelium, but known markers for retinal mosaics are expressed only after the cells have attained a regular spatial arrangement (Wässle and Riemann, 1978;Mitrofanis et al., 1988;Vaney, 1990;C asini and Brecha, 1991;Wikler and Rakic, 1991;Hutsler and Chalupa, 1995;Scheibe et al., 1995), making it difficult to understand how such regularity comes about.Here we report that the transcription factor Islet-1 is an early marker for cholinergic amacrine cells. Islet-1, a member of the LIM homeodomain family known to be involved in vertebrate and invertebrate development (Thor et al...

Exosomes represent an attractive vehicle for the delivery of biomolecules. However, mechanisms for loading functional molecules into exosomes are relatively unexplored. Here we report the use of the evolutionarily conserved late-domain (L-domain) pathway as a mechanism for loading exogenous proteins into exosomes. We demonstrate that labeling of a target protein, Cre recombinase, with a WW tag leads to recognition by the L-domain-containing protein Ndfip1, resulting in ubiquitination and loading into exosomes. Our results show that Ndfip1 expression acts as a molecular switch for exosomal packaging of WW-Cre that can be suppressed using the exosome inhibitor GW4869. When taken up by floxed reporter cells, exosomes containing WW-Cre were capable of inducing DNA recombination, indicating functional delivery of the protein to recipient cells. Engineered exosomes were administered to the brain of transgenic reporter mice using the nasal route to test for intracellular protein delivery in vivo. This resulted in the transport of engineered exosomes predominantly to recipient neurons in a number of brain regions, including the olfactory bulb, cortex, striatum, hippocampus, and cerebellum. The ability to engineer exosomes to deliver biologically active proteins across the blood-brain barrier represents an important step for the development of therapeutics to treat brain diseases.