Contact-FP: A Dimerization-Dependent Fluorescent Protein Toolkit for Visualizing Membrane Contact Site Dynamics.

Membrane contact sites (MCSs) are sites of close apposition between two organelles used to exchange ions, lipids, and information. Cells respond to changing environmental or developmental conditions by modulating the number, extent, or duration of MCSs. Because of their small size and dynamic nature, tools to study the dynamics of MCSs in live cells have been limited. Dimerization-dependent fluorescent proteins (ddFPs) targeted to organelle membranes are an ideal tool for studying MCS dynamics because they reversibly interact to fluoresce specifically at the interface between two organelles. Here, we build on previous work using ddFPs as sensors to visualize the morphology and dynamics of MCSs. We engineered a suite of ddFPs called Contact-FP that targets ddFP monomers to lipid droplets (LDs), the endoplasmic reticulum (ER), mitochondria, peroxisomes, lysosomes, plasma membrane, caveolae, and the cytoplasm. We show that these probes correctly localize to their target organelles. Using LDs as a test case, we demonstrate that Contact-FP pairs specifically localize to the interface between two target organelles. Titration of LD-mitochondria ddFPs revealed that these sensors can be used at high concentrations to drive MCSs or can be titrated down to minimally perturb and visualize endogenous MCSs. We show that Contact-FP probes can be used to: (1) visualize LD-mitochondria MCS dynamics, (2) observe changes in LD-mitochondria MCS dynamics upon overexpression of PLIN5, a known LD-mitochondrial tether, and (3) visualize two MCSs that share one organelle simultaneously (e.g., LD-mitochondria and LD-ER MCSs). Contact-FP probes can be optimized to visualize MCSs between any pair of organelles represented in the toolkit.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy.

Microtubules are polymers of αß-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Atomic force microscopy reveals distinct protofilament-scale structural dynamics in depolymerizing microtubule arrays.

The dynamic reorganization of microtubule-based cellular structures, such as the spindle and the axoneme, fundamentally depends on the dynamics of individual polymers within multimicrotubule arrays. A major class of enzymes implicated in both the complete demolition and fine size control of microtubule-based arrays are depolymerizing kinesins. How different depolymerases differently remodel microtubule arrays is poorly understood. A major technical challenge in addressing this question is that existing optical or electron-microscopy methods lack the spatial-temporal resolution to observe the dynamics of individual microtubules within larger arrays. Here, we use atomic force microscopy (AFM) to image depolymerizing arrays at single-microtubule and protofilament resolution. We discover previously unseen modes of microtubule array destabilization by conserved depolymerases. We find that the kinesin-13 MCAK mediates asynchronous protofilament depolymerization and lattice-defect propagation, whereas the kinesin-8 Kip3p promotes synchronous protofilament depolymerization. Unexpectedly, MCAK can depolymerize the highly stable axonemal doublets, but Kip3p cannot. We propose that distinct protofilament-level activities underlie the functional dichotomy of depolymerases, resulting in either large-scale destabilization or length regulation of microtubule arrays. Our work establishes AFM as a powerful strategy to visualize microtubule dynamics within arrays and reveals how nanometer-scale substrate specificity leads to differential remodeling of micron-scale cytoskeletal structures.

An autoinhibitory clamp of actin assembly constrains and directs synaptic endocytosis.

Synaptic membrane-remodeling events such as endocytosis require force-generating actin assembly. The endocytic machinery that regulates these actin and membrane dynamics localizes at high concentrations to large areas of the presynaptic membrane, but actin assembly and productive endocytosis are far more restricted in space and time. Here we describe a mechanism whereby autoinhibition clamps the presynaptic endocytic machinery to limit actin assembly to discrete functional events. We found that collective interactions between the Drosophila endocytic proteins Nwk/FCHSD2, Dap160/intersectin, and WASp relieve Nwk autoinhibition and promote robust membrane-coupled actin assembly in vitro. Using automated particle tracking to quantify synaptic actin dynamics in vivo, we discovered that Nwk-Dap160 interactions constrain spurious assembly of WASp-dependent actin structures. These interactions also promote synaptic endocytosis, suggesting that autoinhibition both clamps and primes the synaptic endocytic machinery, thereby constraining actin assembly to drive productive membrane remodeling in response to physiological cues.

Neurons constantly talk to each other by sending chemical signals across the tiny gap, or 'synapse', that separates two cells. While inside the emitting cell, these molecules are safely packaged into small, membrane-bound vessels. Upon the right signal, the vesicles fuse with the external membrane of the neuron and spill their contents outside, for the receiving cell to take up and decode. The emitting cell must then replenish its vesicle supply at the synapse through a recycling mechanism known as endocytosis. To do so, it uses dynamically assembling rod-like 'actin' filaments, which work in concert with many other proteins to pull in patches of membrane as new vesicles. The proteins that control endocytosis and actin assembly abound at neuronal synapses, and, when mutated, are linked to many neurological diseases. Unlike other cell types, neurons appear to 'pre-deploy' these actin-assembly proteins to synaptic membranes, but to keep them inactive under normal conditions. How neurons control the way this machinery is recruited and activated remains unknown. To investigate this question, Del Signore et al. conducted two sets of studies. First, they exposed actin to several different purified proteins in initial 'test tube' experiments. This revealed that, depending on the conditions, a group of endocytosis proteins could prevent or promote actin assembly: assembly occurred only if the proteins were associated with membranes. Next, Del Signore et al. mutated these proteins in fruit fly larvae, and performed live cell microscopy to determine their impact on actin assembly and endocytosis. Consistent with the test tube findings, endocytosis mutants had more actin assembly overall, implying that the proteins were required to prevent random actin assembly. However, the same mutants had reduced levels of endocytosis, suggesting that the proteins were also necessary for productive actin assembly. Together, these experiments suggest that, much like a mousetrap holds itself poised ready to spring, some endocytic proteins play a dual role to restrain actin assembly when and where it is not needed, and to promote it at sites of endocytosis. These results shed new light on how neurons might build and maintain effective, working synapses. Del Signore et al. hope that this knowledge may help to better understand and combat neurological diseases, such as Alzheimer's, which are linked to impaired membrane traffic and cell signalling.