The mechanisms of endosome biogenesis and maintenance are largely unknown. The small GTPases Rab 5 and Rab 7 are key determinants of early and late endosomes, organizing effector proteins into specific membrane subdomains. Whether such Rab machineries are indefinitely maintained on membranes or can disassemble in the course of cargo transport is an open question. Here, we combined novel image-analysis algorithms with fast live-cell imaging. We found that the level of Rab 5 dynamically fluctuates on individual early endosomes, linked by fusion and fission events into a network in time. Within it, degradative cargo concentrates in progressively fewer and larger endosomes that migrate from the cell periphery to the center where Rab 5 is rapidly replaced with Rab 7. The class C VPS/HOPS complex, an established GEF for Rab 7, interacts with Rab 5 and is required for Rab 5-to-Rab 7 conversion. Our results reveal unexpected dynamics of Rab domains and suggest Rab conversion as the mechanism of cargo progression between early and late endosomes.

Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.

Particle tracking is of key importance for quantitative analysis of intracellular dynamic processes from time-lapse microscopy image data. Since manually detecting and following large numbers of individual particles is not feasible, automated computational methods have been developed for these tasks by many groups. Aiming to perform an objective comparison of methods, we gathered the community and organized, for the first time, an open competition, in which participating teams applied their own methods independently to a commonly defined data set including diverse scenarios. Performance was assessed using commonly defined measures. Although no single method performed best across all scenarios, the results revealed clear differences between the various approaches, leading to important practical conclusions for users and developers.

The microtubule cytoskeleton is a dynamic structure in which the lengths of the microtubules are tightly regulated. One regulatory mechanism is the depolymerization of microtubules by motor proteins in the kinesin-13 family 1 . These proteins are crucial for the control of microtubule length in cell division [2][3][4] , neuronal development 5 and interphase microtubule dynamics 6,7 . The mechanism by which kinesin-13 proteins depolymerize microtubules is poorly understood. A central question is how these proteins target to microtubule ends at rates exceeding those of standard enzyme-substrate kinetics 8 . To address this question we developed a single-molecule microscopy assay for MCAK, the founding member of the kinesin-13 family 9 . Here we show that MCAK moves along the microtubule lattice in a one-dimensional (1D) random walk. MCAK-microtubule interactions were transient: the average MCAK molecule diffused for 0.83 s with a diffusion coefficient of 0.38 mm 2 s 21 . Although the catalytic depolymerization by MCAK requires the hydrolysis of ATP, we found that the diffusion did not. The transient transition from three-dimensional diffusion to 1D diffusion corresponds to a "reduction in dimensionality" 10 that has been proposed as the search strategy by which DNA enzymes find specific binding sites 11 . We show that MCAK uses this strategy to target to both microtubule ends more rapidly than direct binding from solution.Kinesin-13 motor proteins act at microtubule ends, where they are thought to force protofilaments into a curved conformation 12,13 , which is a likely structural intermediate in the depolymerization process 14 . Classically, kinesin motor proteins reach microtubule ends by ATP-dependent translocation along microtubules. However, LETTERSFigure 1 | MCAK-dependent microtubule depolymerization. a, Diagram of the in vitro assay depicting a microtubule (red) immobilized above the glass surface by anti-tubulin antibodies (dark blue). Excitation by total internal reflection allows the detection of single molecules (namely MCAK-GFP in green) in the evanescent field (shown in blue). b, Epifluorescence images of immobilized microtubules at the times shown in minutes. MCAK dimer (8 nM) was added at t ¼ 2 min. c, Plot of microtubule depolymerization rate against MCAK concentration. Error bars are s.d. Data fitted to Hill equations (lines plotted) yielded K m ¼ 3.9 nM and K m ¼ 6.1 nM for MCAK and MCAK-GFP, and n ¼ 2.4 and n ¼ 2.2, respectively. Red squares, MCAK-His 6 ; green circles, MCAK-His 6 -EGFP. d, Shortening of four microtubules from a mean length of 8.4 mm to 7.8 mm (black line) after the addition of 5 nM MCAK (red line). The depolymerization rate approached steady state with a time constant of 3.8 s (green fitted line). (Fig. 1a) in which microtubules were immobilized on coverslips by means of surface-adsorbed anti-tubulin antibodies. Individual rhodamine-labelled microtubules and single MCAK-GFP molecules were revealed by epifluorescence and total-internalreflection fluorescence (TIRF) illumina...

In the developing fly wing, secreted morphogens such as Decapentaplegic (Dpp) and Wingless (Wg) form gradients of concentration providing positional information. Dpp forms a longer-range gradient than Wg. To understand how the range is controlled, we measured the four key kinetic parameters governing morphogen spreading: the production rate, the effective diffusion coefficient, the degradation rate, and the immobile fraction. The four parameters had different values for Dpp versus Wg. In addition, Dynamin-dependent endocytosis was required for spreading of Dpp, but not Wg. Thus, the cellular mechanisms of Dpp and Wingless spreading are different: Dpp spreading requires endocytic, intracellular trafficking.

Endocytosis is a complex process fulfilling many cellular and developmental functions. Understanding how it is regulated and integrated with other cellular processes requires a comprehensive analysis of its molecular constituents and general design principles. Here, we developed a new strategy to phenotypically profile the human genome with respect to transferrin (TF) and epidermal growth factor (EGF) endocytosis by combining RNA interference, automated high-resolution confocal microscopy, quantitative multiparametric image analysis and high-performance computing. We identified several novel components of endocytic trafficking, including genes implicated in human diseases. We found that signalling pathways such as Wnt, integrin/cell adhesion, transforming growth factor (TGF)-beta and Notch regulate the endocytic system, and identified new genes involved in cargo sorting to a subset of signalling endosomes. A systems analysis by Bayesian networks further showed that the number, size, concentration of cargo and intracellular position of endosomes are not determined randomly but are subject to specific regulation, thus uncovering novel properties of the endocytic system.

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

Rab GTPases and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are evolutionarily conserved essential components of the eukaryotic intracellular transport system. Although pairing of cognate SNAREs is sufficient to fuse membranes in vitro, a complete reconstitution of the Rab-SNARE machinery has never been achieved. Here we report the reconstitution of the early endosomal canine Rab5 GTPase, its key regulators and effectors together with SNAREs into proteoliposomes using a set of 17 recombinant human proteins. These vesicles behave like minimal 'synthetic' endosomes, fusing with purified early endosomes or with each other in vitro. Membrane fusion measured by content-mixing and morphological assays requires the cooperativity between Rab5 effectors and cognate SNAREs which, together, form a more efficient 'core machinery' than SNAREs alone. In reconstituting a fusion mechanism dependent on both a Rab GTPase and SNAREs, our work shows that the two machineries act coordinately to increase the specificity and efficiency of the membrane tethering and fusion process.