Microtubules are polarized polymers that exhibit dynamic instability, with alternating phases of elongation and shortening, particularly at the more dynamic plus-end. Microtubule plus-end tracking proteins (؉TIPs) localize to and track with growing microtubule plus-ends in the cell. ؉TIPs regulate microtubule dynamics and mediate interactions with other cellular components. The molecular mechanisms responsible for the ؉TIP tracking activity are not well understood, however. We reconstituted the ؉TIP tracking of mammalian proteins EB1 and CLIP-170 in vitro at single-molecule resolution using time-lapse total internal reflection fluorescence microscopy. We found that EB1 is capable of dynamically tracking growing microtubule plus-ends. Our singlemolecule studies demonstrate that EB1 exchanges rapidly at microtubule plus-ends with a dwell time of <1 s, indicating that single EB1 molecules go through multiple rounds of binding and dissociation during microtubule polymerization. CLIP-170 exhibits lattice diffusion and fails to selectively track microtubule ends in the absence of EB1; the addition of EB1 is both necessary and sufficient to mediate plus-end tracking by CLIP-170. Single-molecule analysis of the CLIP-170 -EB1 complex also indicates a short dwell time at growing plus-ends, an observation inconsistent with the copolymerization of this complex with tubulin for plus-end-specific localization. GTP hydrolysis is required for ؉TIP tracking, because end-specificity is lost when tubulin is polymerized in the presence of guanosine 5-[␣,␤-methylene]triphosphate (GMPCPP). Together, our data provide insight into the mechanisms driving plus-end tracking by mammalian ؉TIPs and suggest that EB1 specifically recognizes the distinct lattice structure at the growing microtubule end.ϩTIP ͉ single molecule ͉ total internal reflection fluorescence (TIRF) microscopy ͉ dynamic instability

Long-range retrograde axonal transport in neurons is driven exclusively by the microtubule motor cytoplasmic dynein. The efficient initiation of dynein-mediated transport from the distal axon is critical for normal neuronal function, and neurodegenerative diseaseassociated mutations have been shown to specifically disrupt this process. Here, we examine the role of dynamic microtubules and microtubule plus-end binding proteins (ϩTIPs) in the initiation of dynein-mediated retrograde axonal transport using live-cell imaging of cargo motility in primary mouse dorsal root ganglion neurons. We show that end-binding (EB)-positive dynamic microtubules are enriched in the distal axon. The ϩTIPs EB1, EB3, and cytoplasmic linker protein-170 (CLIP-170) interact with these dynamic microtubules, recruiting the dynein activator dynactin in an ordered pathway, leading to the initiation of retrograde transport by the motor dynein. Once transport has initiated, however, neither the EBs nor CLIP-170 are required to maintain transport flux along the mid-axon. In contrast, the ϩTIP Lis1 activates transport through a distinct mechanism and is required to maintain processive organelle transport along both the distal and mid-axon. Further, we show that the EB/CLIP-170/dynactin-dependent mechanism is required for the efficient initiation of transport from the distal axon for multiple distinct cargos, including mitochondria, Rab5-positive early endosomes, late endosomes/lysosomes, and TrkA-, TrkB-, and APP-positive organelles. Our observations indicate that there is an essential role for ϩTIPs in the regulation of retrograde transport initiation in the neuron.

Summary Motor-cargo recruitment to microtubules is often the rate-limiting step of intracellular transport, and defects in this recruitment can cause neurodegenerative disease. Here, we use in vitro reconstitution assays with single molecule resolution, live-cell transport assays in primary neurons, computational image analysis and computer simulations to investigate the factors regulating retrograde transport initiation in the distal axon. We find that phosphorylation of the cytoskeletal-organelle linker protein CLIP-170 and post-translational modifications of the microtubule track combine to precisely control the initiation of retrograde transport. Computer simulations of organelle dynamics in the distal axon indicate that while CLIP-170 primarily regulates the time to microtubule encounter, the tyrosination state of the microtubule lattice regulates the likelihood of binding. These mechanisms interact to control transport initiation in the axon in a manner sensitive to the specialized cytoskeletal architecture of the neuron.

Summary Microtubules undergo alternating periods of growth and shortening, known as dynamic instability. These dynamics allow microtubule plus ends to explore cellular space. The ‘search-and-capture’ model posits that selective anchoring of microtubule plus ends at the cell cortex may contribute to cell polarization, spindle orientation, or targeted trafficking to specific cellular domains [1-3]. While cytoplasmic dynein is primarily known as a minus end-directed microtubule motor for organelle transport, cortically-localized dynein has been shown to capture and tether microtubules at the cell periphery in both dividing and interphase cells [3-7]. To explore the mechanism involved, we developed a minimal in vitro system, with dynein-bound beads positioned near microtubule plus-ends using an optical trap. Dynein induced a significant reduction in the lateral diffusion of microtubule ends, distinct from the effects of other microtubule-associated proteins such as kinesin-1 and EB1. In assays with dynamic microtubules, dynein delayed barrier-induced catastrophe of microtubules. This effect was ATP-dependent, indicating that dynein motor activity was required. Computational modelling suggests that dynein delays catastrophe by exerting tension on individual protofilaments leading to microtubule stabilization. Thus, dynein-mediated capture and tethering of microtubules at the cortex can lead to enhanced stability of dynamic plus-ends.

The dynein partner dynactin not only binds to microtubules, but is found to potently influence microtubule dynamics in neurons.