Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.

Targeted manipulation of activity in specific populations of neurons is important for investigating the neural circuit basis of behavior. Optogenetic approaches using light-sensitive microbial rhodopsins have permitted manipulations to reach a level of temporal precision that is enabling functional circuit dissection. As demand for more precise perturbations to serve specific experimental goals increases, a palette of opsins with diverse selectivity, kinetics, and spectral properties will be needed. Here, we introduce a novel approach of "topological engineering"-inversion of opsins in the plasma membrane-and demonstrate that it can produce variants with unique functional properties of interest for circuit neuroscience. In one striking example, inversion of a Channelrhodopsin variant converted it from a potent activator into a fast-acting inhibitor that operates as a cation pump. Our findings argue that membrane topology provides a useful orthogonal dimension of protein engineering that immediately permits as much as a doubling of the available toolkit.

In optical microscopy, fine structural details are resolved by using refraction to magnify images of a specimen. We discovered that by synthesizing a swellable polymer network within a specimen, it can be physically expanded, resulting in physical magnification. By covalently anchoring specific labels located within the specimen directly to the polymer network, labels spaced closer than the optical diffraction limit can be isotropically separated and optically resolved, a process we call expansion microscopy (ExM). Thus, this process can be used to perform scalable superresolution microscopy with diffraction-limited microscopes. We demonstrate ExM with apparent ~70-nanometer lateral resolution in both cultured cells and brain tissue, performing three-color superresolution imaging of ~107 cubic micrometers of the mouse hippocampus with a conventional confocal microscope.

Expansion microscopy (ExM) is a recently developed technique that enables nanoscale-resolution imaging of preserved cells and tissues on conventional diffraction-limited microscopes via isotropic physical expansion of the specimens before imaging. In ExM, biomolecules and/or fluorescent labels in the specimen are linked to a dense, expandable polymer matrix synthesized evenly throughout the specimen, which undergoes 3-dimensional expansion by ∼4.5 fold linearly when immersed in water. Since our first report, versions of ExM optimized for visualization of proteins, RNA, and other biomolecules have emerged. Here we describe best-practice, step-by-step ExM protocols for performing analysis of proteins (protein retention ExM, or proExM) as well as RNAs (expansion fluorescence in situ hybridization, or ExFISH), using chemicals and hardware found in a typical biology lab. Furthermore, a detailed protocol for handling and mounting expanded samples and for imaging them with confocal and light-sheet microscopes is provided. © 2018 by John Wiley & Sons, Inc.

Expansion microscopy (ExM) is a physical form of magnification that increases the effective resolving power of any microscope. Here, we describe the fundamental principles of ExM, as well as how recently developed ExM variants build upon and apply those principles. We examine applications of ExM in cell and developmental biology for the study of nanoscale structures as well as ExM's potential for scalable mapping of nanoscale structures across large sample volumes. Finally, we explore how the unique anchoring and hydrogel embedding properties enable postexpansion molecular interrogation in a purified chemical environment. ExM promises to play an important role complementary to emerging live-cell imaging techniques, because of its relative ease of adoption and modification and its compatibility with tissue specimens up to at least 200 μm thick. Expected final online publication date for the , Volume 35 is October 7, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Expansion microscopy (ExM) is an innovative approach to achieve super-resolution images without using super-resolution microscopes, based on the physical expansion of the sample. The advent of ExM has unlocked super-resolution imaging for a broader scientific circle, lowering the cost and entry skill requirements to the field. One of its branches, ultrastructure ExM (U-ExM), has become popular among research groups studying Apicomplexan parasites, including the acute stage of Toxoplasma gondii infection. The chronic cyst-forming stage of Toxoplasma, however, resists U-ExM expansion, impeding precise protein localisation. Here, we solve the in vitro cyst’s resistance to denaturation required for successful U-ExM of the encapsulated parasites. As the cyst’s main structural protein CST1 contains a mucin domain, we added an enzymatic digestion step using the pan-mucinase StcE prior to the expansion protocol. This allowed full expansion of the cysts in fibroblasts and primary neuronal cell culture without interference with the epitopes of the cyst-wall associated proteins. Using StcE-enhanced U-ExM, we clarified the shape and location of the GRA2 protein important for establishing a normal cyst. Expanded cysts revealed GRA2 granules spanning across the cyst wall, with a notable presence observed outside on both sides of the CST1-positive layer.

Importance Toxoplasma gondii is an intracellular parasite capable of establishing long-term chronic infection in nearly all warm-blooded animals. During the chronic stage, parasites encapsulate into cysts in a wide range of tissues but particularly in neurons of the central nervous system and in skeletal muscle. Current anti-Toxoplasma drugs do not eradicate chronic parasites and leave behind a reservoir of infection. As the cyst is critical for both transmission and pathology of the disease, we need to understand more fully the biology of the cyst and its vulnerabilities.

The advent of a new super-resolution approach called ultrastructure expansion microscopy allowed in-depth studies of the acute stage of Toxoplasma infection but not the cyst-forming stage, which resists protocol-specific denaturation. Here, we show that an additional step of enzymatic digestion using mucinase StcE allows full expansion of the Toxoplasma cysts, offering a new avenue for a comprehensive examination of the chronic stage of infection using an accessible super-resolution technique.

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 µm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine novel spatially and molecularly defined subregions. EASI-FISH also facilitates iterative re-analysis of scRNA-Seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

The excitability of individual dendritic branches is a plastic property of neurons. We found that experience in an enriched environment increased propagation of dendritic Na(+) spikes in a subset of individual dendritic branches in rat hippocampal CA1 pyramidal neurons and that this effect was mainly mediated by localized downregulation of A-type K(+) channel function. Thus, dendritic plasticity might be used to store recent experience in individual branches of the dendritic arbor.

The hippocampus is critical for producing stable representations of familiar spaces. How these representations arise is poorly understood, largely because changes to hippocampal inputs have not been measured during spatial learning. Here, using intracellular recording, we monitored inputs and plasticity-inducing complex spikes (CSs) in CA1 neurons while mice explored novel and familiar virtual environments. Inputs driving place field spiking increased in amplitude - often suddenly - during novel environment exploration. However, these increases were not sustained in familiar environments. Rather, the spatial tuning of inputs became increasingly similar across repeated traversals of the environment with experience - both within fields and throughout the whole environment. In novel environments, CSs were not necessary for place field formation. Our findings support a model in which initial inhomogeneities in inputs are amplified to produce robust place field activity, then plasticity refines this representation into one with less strongly modulated, but more stable, inputs for long-term storage.

Synaptic plasticity in adult neural circuits may involve the strengthening or weakening of existing synapses as well as structural plasticity, including synapse formation and elimination. Indeed, long-term in vivo imaging studies are beginning to reveal the structural dynamics of neocortical neurons in the normal and injured adult brain. Although the overall cell-specific morphology of axons and dendrites, as well as of a subpopulation of small synaptic structures, are remarkably stable, there is increasing evidence that experience-dependent plasticity of specific circuits in the somatosensory and visual cortex involves cell type-specific structural plasticity: some boutons and dendritic spines appear and disappear, accompanied by synapse formation and elimination, respectively. This Review focuses on recent evidence for such structural forms of synaptic plasticity in the mammalian cortex and outlines open questions.