The fast turnover of membrane components through endocytosis and recycling allows precise control of the composition of the plasma membrane. Endocytic recycling can be rapid with some molecules returning to the plasma membrane with a <5 minutes. Existing methods to study these trafficking pathways utilize chemical, radioactive, or fluorescent labeling of cell surface receptors in pulse-chase experiments, which require tedious washing steps and manual collection of samples. Here, we introduce a live-cell endocytic recycling assay, based on a newly designed cell-impermeable, fluorogenic ligand for HaloTag: 'Janelia Fluor 635i' (JFi; i=impermeant) which allows real-time detection of membrane receptor recycling at steady state. We used this method to study the effect of iron depletion on transferrin receptor (TfR) recycling using the chelator desferrioxamine. We found this perturbation significantly increases the TfR recycling rate. The high temporal resolution and simplicity of this assay provides a clear advantage over extant methods and makes it ideal for large scale cellular imaging studies. This assay can be adapted to examine other cellular kinetic parameters such as protein turnover and biosynthetic trafficking.

To study the complex synaptic interactions underpinning dendritic information processing in single neurons, experimenters require methods to mimic presynaptic neurotransmitter release at multiple sites with no physiological damage. We show that laser scanning systems built around large-aperture acousto-optic deflectors and high numerical aperture objective lenses provide the sub-millisecond, sub-micron precision necessary to achieve physiological, exogenous synaptic stimulation. Our laser scanning systems can produce the sophisticated spatio-temporal patterns of synaptic input that are necessary to investigate single-neuron dendritic physiology.

Symmetric cell division requires the even partitioning of genetic information and cytoplasmic contents between daughter cells. Whereas the mechanisms coordinating the segregation of the genome are well known, the processes that ensure organelle segregation between daughter cells remain less well understood. Here we identify multiple actin assemblies with distinct but complementary roles in mitochondrial organization and inheritance in mitosis. First, we find a dense meshwork of subcortical actin cables assembled throughout the mitotic cytoplasm. This network scaffolds the endoplasmic reticulum and organizes three-dimensional mitochondrial positioning to ensure the equal segregation of mitochondrial mass at cytokinesis. Second, we identify a dynamic wave of actin filaments reversibly assembling on the surface of mitochondria during mitosis. Mitochondria sampled by this wave are enveloped within actin clouds that can spontaneously break symmetry to form elongated comet tails. Mitochondrial comet tails promote randomly directed bursts of movement that shuffle mitochondrial position within the mother cell to randomize inheritance of healthy and damaged mitochondria between daughter cells. Thus, parallel mechanisms mediated by the actin cytoskeleton ensure both equal and random inheritance of mitochondria in symmetrically dividing cells.

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are challenging to detect with fluorescence microscopy, making it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed fluorescent-protein-tagged actin nanobodies, termed 'actin chromobodies' (ACs), targeted to organelle membranes to enable high-resolution imaging of sub-organellar actin dynamics.

Cytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1 min, cortical actin reduced across the synapse, T cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5 min and reached the synapse after 6 min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion.

The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterized the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy (iPALM), a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy (SIM) and localization-based superresolution microscopy. Phagocytic podosomes were observed during frustrated phagocytosis, a model in which cells attempt to engulf micro-patterned IgG antibodies. For circular patterns, this resulted in regular arrays of podosomes with well-defined geometry. Using persistent homology, we developed a pipeline for semi-automatic identification and measurement of podosome features. These studies revealed an "hourglass" shape of the podosome actin core, a protruding "knob" at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, α-actinin, cortactin, and microtubules relative to actin were characterized.

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

Subiculum is the primary output area of the hippocampus and serves as a key relay center in the process of memory formation and retrieval. A majority of subicular pyramidal neurons communicate via bursts of action potentials, a mode of signaling that may enhance the fidelity of information transfer and synaptic plasticity or contribute to epilepsy when unchecked. In the present study, we show that a Ca(2+) tail current drives bursting in subicular pyramidal neurons. An action potential activates voltage-activated Ca(2+) channels, which deactivate slowly enough during action potential repolarization to produce an afterdepolarization that triggers subsequent action potentials in the burst. The Ca(2+) channels underlying bursting are located primarily near the soma, and the amplitude of Ca(2+) tail currents correlates with the strength of bursting across cells. Multiple channel subtypes contribute to Ca(2+) tail current, but the need for an action potential to produce the slow depolarization suggests a central role for high-voltage-activated Ca(2+) channels in subicular neuron bursting.