Dopaminergic signaling and plasticity are essential to numerous central nervous system functions and pathologies, including movement, cognition and addiction. The amphetamine-and cocaine-sensitive dopamine (DA) transporter (DAT) tightly controls extracellular DA concentrations and half-life. DAT function and surface expression are not static, but are dynamically modulated by membrane trafficking. We recently demonstrated that the DAT carboxy terminus encodes a PKC-sensitive internalization signal that also suppresses basal DAT endocytosis. However, the cellular machinery governing regulated DAT trafficking is not well defined. In work presented here, we identified the Ras-like GTPase, Rin (Rit2) as a protein that interacts with the DAT carboxy terminal endocytic signal. Yeast two-hybrid, GST pulldown and FRET studies establish that DAT and Rin directly interact, and co-localization studies reveal that DAT/Rin associations occur primarily in lipid raft microdomains. Co-immunoprecipitations demonstrate that PKC activation regulates Rin association with DAT. Perturbation of Rin function with GTPase mutants and shRNA-mediated Rin knockdown reveals that Rin is critical for PKC-mediated DAT internalization and functional downregulation. These results establish that Rin is a DAT-interacting protein that is required for PKC-regulated DAT trafficking. Moreover, this work suggests that Rin participates in regulated endocytosis.

Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic.endosomal sorting | lysosomal degradation | super resolution | receptor trafficking | patterned illumination T he endocytic pathway is known principally for its role in the uptake of receptors and ligands (1-4) and increasingly is being recognized as critical in other aspects of cell physiology. For example, the control of cellular growth through mammalian target of rapamycin signaling involves associations with late endosomal membranes (5, 6), and microRNA regulatory mechanisms are associated with endosomal/lysosomal membranes (7-9). Thus, it is necessary to understand how the distinct membrane compartments of the endocytic pathway form, traffic internalized ligands, and serve as scaffolds for the assembly of regulatory complexes.In the classical view of the endocytic pathway (3, 10-13), internalizing cargoes are incorporated into vesicles formed by clathrin-mediated or other mechanisms of endocytosis at the plasma membrane. These vesicles fuse with a homogenous population of early endosomes from which cargo then is sorted to different destinations. However, recent results demonstrate that genetic networks involved in the early trafficking of the transferrin (Tf) and EGF receptors (TfR and EGFR, respectively) differ from each other substantially (14) and find that EGF and Tf populate different subsets of vesicles almost immediately after addition to live cells at physiological temperature (15). Further evidence for early endosome heterogeneity is found in studies in which endosomes display different motilities and different phosphoinositide effector complements (16-18). These results suggest that different populations of e...

siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs®) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention.

Dopamine (DA) reuptake terminates dopaminergic neurotransmission and is mediated by DA transporters (DATs). Acute protein kinase C (PKC) activation accelerates DAT internalization rates, thereby reducing DAT surface expression. Basal DAT endocytosis and PKC-stimulated DAT functional downregulation rely on residues within the 587-596 region, although whether PKCinduced DAT downregulation reflects transporter endocytosis mechanisms linked to those controlling basal endocytosis rates is unknown. Here, we define residues governing basal and PKCstimulated DAT endocytosis. Alanine substituting DAT residues 587-590 1) abolished PKC stimulation of DAT endocytosis, and 2) markedly accelerated basal DAT internalization, comparable to that of wildtype DAT during PKC activation. Accelerated basal DAT internalization relied specifically on residues 588-590, which are highly conserved among SLC6 neurotransmitter transporters. Our results support a model whereby residues within the 587-590 stretch may serve as a locus for a PKC-sensitive braking mechanism that tempers basal DAT internalization rates.

Amphetamine (AMPH) is a potent dopamine (DA) transporter (DAT) inhibitor that markedly increases extracellular DA levels. In addition to its actions as a DAT antagonist, acute AMPH exposure induces DAT losses from the plasma membrane, implicating transporter-specific membrane trafficking in amphetamine\u27s actions. Despite reports that AMPH modulates DAT surface expression, the trafficking mechanisms leading to this effect are currently not defined. We recently reported that DAT residues 587-596 play an integral role in constitutive and protein kinase C (PKC)-accelerated DAT internalization. In the current study, we tested whether the structural determinants required for PKC-stimulated DAT internalization are necessary for AMPH-induced DAT sequestration. Acute amphetamine exposure increased DAT endocytic rates, but DAT carboxy terminal residues 587-590, which are required for PKC-stimulated internalization, were not required for AMPH-accelerated DAT endocytosis. AMPH decreased DAT endocytic recycling, but did not modulate transferrin receptor recycling, suggesting that AMPH does not globally diminish endocytic recycling. Finally, treatment with a PKC inhibitor demonstrated that AMPH-induced DAT losses from the plasma membrane were not dependent upon PKC activity. These results suggest that the mechanisms responsible for AMPH-mediated DAT internalization are independent from those governing PKC-sensitive DAT endocytosis

hypertranslocation ͉ hybrid ͉ stability ͉ topological lock T ranscription lies at the heart of cellular gene expression. Unlike replication, transcription is not distributive; that is, any dissociation of an elongation complex is a terminal event. Consequently, elongation complexes must be highly stable while transcribing at up to several hundred bases per second. Recent studies demonstrate, however, that elongation is not a uniform process, with reasonably long-lived, sequence-dependent pauses occurring stochastically (1-4). Indeed, the elongation phase is well known to be subject to regulation through attenuation: sequence-dependent signals leading to pause, arrest, slippage, or termination (5). Elongating RNA polymerases must hold the DNA template and the RNA product tightly enough to be highly stable through nonterminating pauses, yet they must be able to dissociate the complex in response to specific sequences in the DNA. Understanding this interplay requires understanding mechanisms of dissociation.In classic rho-independent or ''intrinsic'' termination, dissociation is thought to occur as the polymerase slows in response to sequence, and structure begins to form concurrently in the nascent transcript (6-8). A run of encoded U's has been proposed both to slow transcription and to weaken the RNA-DNA hybrid. Recently, it has been proposed that, rather than direct dissociation of the nascent transcript from a complex halted at the primary termination site (with or without allosteric assistance), dissociation occurs primarily from forward-translocated states (9-11). In forwardtranslocated (hypertranslocated) states, the hybrid is shortened, weakening the binding of the RNA to the complex. Additionally, shortening of the hybrid may also serve to remove a topological locking of the RNA onto the template strand. As illustrated in Fig. 1, dissociation of the RNA transcript would be substantially faster from forward translocated states than from the initially paused state. Although forward-translocated states are expected to be energetically disfavored relative to the initial on-pathway state and so would be expected to exist at lower populations, the difference in the kinetics of dissociation could compensate such that the dominant dissociative pathway proceeds via forward translocated states.The model shown in Fig. 1 predicts certain behaviors. In general, forward translocation requires melting of the DNA duplex downstream and dissociation of the most upstream base(s) in the hybrid. These costs are partially offset by the energy gained by collapse of a base pair in the DNA at the upstream edge of the bubble. Thus, one would expect that altering the balance between these effects could shift the distribution toward more forward-translocated states and therefore toward or away from dissociation. As demonstrated previously, a direct crosslink between the DNA strands downstream of the halt site should prevent forward translocation and is in fact seen to reduce termination from an intrinsic terminator (9).We propose t...

Recent work has led to the identification of novel endocytic compartments with functional roles in both protein trafficking and growth factor signal transduction. The phosphatidylinositol 3-phosphate binding, FYVE domain-containing protein WDFY2 is localized to a distinct subset of early endosomes, which are localized close to the plasma membrane. Here, we find that the serine/threonine kinase Akt interacts with these endosomes in an isoform-specific manner. Using quantitative fluorescence microscopy we demonstrate specific co-localization of WDFY2 with endogenous Akt2, but not Akt1. Moreover, depletion of WDFY2 leads to impaired phosphorylation of Akt in response to insulin due to isoform specific reduction of Akt2, but not Akt1, protein levels, and to a marked reduction in the insulin-stimulated phosphorylation of numerous Akt substrates. This is accompanied by an impairment in insulin-stimulated glucose transport and, after prolonged silencing, a reduction in the level of expression of adipogenic genes. We propose that WDFY2-enriched endosomes serve as a scaffold that enables specificity of insulin signaling through Akt2.The early endocytic pathway is increasingly being recognized as a complex and heterogeneous membrane population in which distinct endosomal populations are specialized for the trafficking of different receptor types (1, 2). Complexity and specialization in the endosomal pathway are achieved by the action of small GTPases and by the generation of specific phosphoinositides on the endosomal surface. One of the best studied examples of this mechanism is the specific and temporal targeting of proteins containing FYVE domains to phosphatidylinositol 3-phosphate (3-6), which is present almost exclusively in endosomal membranes. The human genome encodes for Ͼ30 proteins that contain FYVE domains, several of which are highly conserved and which may contribute in different ways toward establishing the complexity and functionality of the endocytic pathway. We recently characterized one of these proteins, WDFY2, named for its content of WD40 motifs and a FYVE domain (7). In Caenorhabditis elegans, WDFY2 depletion impairs endocytosis in coelomocytes, and in mammalian cells it defines a distinct set of endosomes that lack the canonical markers EEA1 and Rab5 and are further distinguished by their close proximity to the plasma membrane (7,8).In addition to internalization, the endosomal pathway plays a critical role in modulating signal transduction. Growth factor receptors are internalized immediately following activation, and both their fate and their signaling functions are affected by their transit through the endocytic pathway (9 -13). Different receptors traffic through distinct early endosomal compartments (1, 2), and their signaling functions are modulated by the specific nature of the endosomes through which they traffic. For example, signaling by transforming growth factor ␤ is influenced by the endosomal localization of the SMAD-interacting protein SARA, which is found in endosomes containing the...

The ability to isolate and subsequently culture mitotically active female germ cells from adult ovaries, referred to as either oogonial stem cells (OSCs) or adult female germline stem cells (aFGSCs), has provided a robust system to study female germ cell development under multiple experimental conditions, and in many species. Flow cytometry or fluorescence-activated cell sorting (FACS) is an integral part of many isolation and characterization protocols. Here, we provide methodological details for antibody-based flow cytometric isolation of OSCs using antibodies specific for external epitopes of the proteins Ddx4 or Ifitm3, alone or in combination with the use of fluorescent reporter mice. Beginning with sample preparation, we provide point-by-point instructions to guide researchers on how to isolate OSCs using flow cytometry.