Dinah Loerke

Bio: Dinah Loerke is an academic researcher from University of Denver. The author has contributed to research in topics: Clathrin & Endocytic cycle. The author has an hindex of 20, co-authored 32 publications receiving 3030 citations. Previous affiliations of Dinah Loerke include Scripps Research Institute & Max Planck Society.

Robust single-particle tracking in live-cell time-lapse sequences.

Abstract: Single-particle tracking (SPT) is often the rate-limiting step in live-cell imaging studies of subcellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose solution identifies the overall most likely set of particle trajectories throughout a movie. Using this approach, we show that the GTPase dynamin differentially affects the kinetics of long- and short-lived endocytic structures and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.

Cargo and dynamin regulate clathrin-coated pit maturation.

Abstract: Total internal reflection fluorescence microscopy (TIR-FM) has become a powerful tool for studying clathrin-mediated endocytosis. However, due to difficulties in tracking and quantifying their heterogeneous dynamic behavior, detailed analyses have been restricted to a limited number of selected clathrin-coated pits (CCPs). To identify intermediates in the formation of clathrin-coated vesicles and factors that regulate progression through these stages, we used particle-tracking software and statistical methods to establish an unbiased and complete inventory of all visible CCP trajectories. We identified three dynamically distinct CCP subpopulations: two short-lived subpopulations corresponding to aborted intermediates, and one longer-lived productive subpopulation. In a manner dependent on AP2 adaptor complexes, increasing cargo concentration significantly enhances the maturation efficiency of productive CCPs, but has only minor effects on their lifetimes. In contrast, small interfering RNA (siRNA) depletion of dynamin-2 GTPase and reintroduction of wild-type or mutant dynamin-1 revealed dynamin's role in controlling the turnover of abortive intermediates and the rate of CCP maturation. From these data, we infer the existence of an endocytic restriction or checkpoint, responsive to cargo and regulated by dynamin.

The last few milliseconds in the life of a secretory granule

Abstract: We have monitored single vesicles (granules) in bovine adrenal chromaffin cells using an optical sectioning technique, total internal reflection fluorescence microscopy (TIRFM). With TIR, fluorescence excitation is limited to an optical slice near a glass/water interface. In cells located at the interface, granules loaded with fluorescent dye can be visualized near to or docked at the plasma membrane. Here we give evidence that (1) TIRFM resolves single vesicles and (2) the fluorescence signal originates from vesicles of roughly 350 nm diameter, presumably large dense core vesicles (LDCVs). (3) Diffusional spread of released vesicle contents can be resolved and serves as a convenient criterion for a fusion event. (4) We give details on vesicle properties in resting cells, such as lateral mobility of chromaffin granules, number density, and frequency of spontaneous fusion or withdrawal into the cytoplasm. (5) Upon stimulation with high extracellular potassium, TIRFM reports depletion of the `visible pool' of vesicles closest to the plasma membrane within hundreds of milliseconds, consistent with previous concepts of a release-ready pool. We conclude that TIRFM constitutes an independent assay for pool depletion. TIRFM will allow us to study aspects of secretion that have previously been inaccessible in living cells, in particular the spatial relations and dynamics of vesicles prior to and during exocytosis and re-supply of the near-membrane pool of vesicles.

Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis

Abstract: Clathrin-mediated endocytosis of surface receptors and their bound ligands (i.e., cargo) is highly regulated, including by the cargo itself. One of the possible sources of the observed heterogeneous dynamics of clathrin-coated pits (CCPs) might be the different cargo content. Consistent with this, we show that CCP size and dynamic behavior varies with low density lipoprotein receptor (LDLR) expression levels in a manner dependent on the LDLR-specific adaptors, Dab2 and ARH. In Dab2-mCherry–expressing cells, varying LDLR expression leads to a progressive increase in CCP size and to the appearance of nonterminal endocytic events. In LDLR and ARH-mCherry–expressing cells in addition to an increase in CCP size, turnover of abortive CCPs increases, and the rate of CCP maturation decreases. Altogether, our results underscore the highly dynamic and cargo-responsive nature of CCP assembly and suggest that the observed heterogeneity is, in part, related to compositional differences (e.g., cargo and adaptors) between CCPs.

Regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of AP2.

Abstract: The critical initiation phase of clathrin-mediated endocytosis (CME) determines where and when endocytosis occurs. Heterotetrameric adaptor protein 2 (AP2) complexes, which initiate clathrin-coated pit (CCP) assembly, are activated by conformational changes in response to phosphatidylinositol-4,5-bisphosphate (PIP2) and cargo binding at multiple sites. However, the functional hierarchy of interactions and how these conformational changes relate to distinct steps in CCP formation in living cells remains unknown. We used quantitative live-cell analyses to measure discrete early stages of CME and show how sequential, allosterically regulated conformational changes activate AP2 to drive both nucleation and subsequent stabilization of nascent CCPs. Our data establish that cargoes containing Yxxφ motif, but not dileucine motif, play a critical role in the earliest stages of AP2 activation and CCP nucleation. Interestingly, these cargo and PIP2 interactions are not conserved in yeast. Thus, we speculate that AP2 has evolved as a key regulatory node to coordinate CCP formation and cargo sorting and ensure high spatial and temporal regulation of CME.

Molecular mechanism and physiological functions of clathrin-mediated endocytosis

Abstract: Clathrin-mediated endocytosis is the endocytic portal into cells through which cargo is packaged into vesicles with the aid of a clathrin coat. It is fundamental to neurotransmission, signal transduction and the regulation of many plasma membrane activities and is thus essential to higher eukaryotic life. Morphological stages of vesicle formation are mirrored by progression through various protein modules (complexes). The process involves the formation of a putative FCH domain only (FCHO) initiation complex, which matures through adaptor protein 2 (AP2)-dependent cargo selection, and subsequent coat building, dynamin-mediated scission and finally auxilin- and heat shock cognate 70 (HSC70)-dependent uncoating. Some modules can be used in other pathways, and additions or substitutions confer cell specificity and adaptability.

Methods for cell and particle tracking.

Abstract: Achieving complete understanding of any living thing inevitably requires thorough analysis of both its anatomic and dynamic properties. Live-cell imaging experiments carried out to this end often produce massive amounts of time-lapse image data containing far more information than can be digested by a human observer. Computerized image analysis offers the potential to take full advantage of available data in an efficient and reproducible manner. A recurring task in many experiments is the tracking of large numbers of cells or particles and the analysis of their (morpho)dynamic behavior. In the past decade, many methods have been developed for this purpose, and software tools based on these are increasingly becoming available. Here, we survey the latest developments in this area and discuss the various computational approaches, software tools, and quantitative measures for tracking and motion analysis of cells and particles in time-lapse microscopy images.

Membrane Fusion and Exocytosis

Abstract: ▪ Abstract Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intr...