The endoplasmic reticulum (ER) is an important regulator of Ca2+ in cells and dysregulation of ER calcium homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impacts cellular physiology would likely be facilitated by more quantitative measurements of ER Ca2+ levels that allow easier comparisons across conditions. Here, we developed a ratiometric version of our original ER-GCaMP probe that allows for more quantitative comparisons of the concentration of Ca2+ in the ER across cell types and sub-cellular compartments. Using this approach we show that the resting concentration of ER Ca2+ in primary dissociated neurons is substantially lower than that in measured in embryonic fibroblasts.

Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.

Intracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry (MS)-based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a green fluorescent protein (GFP)-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose-dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by MS and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high-throughput applications of variables that affect aspartate levels.

Behavior relies on activity in structured neural circuits that are distributed across the brain, but most experiments probe neurons in a single area at a time. Using multiple Neuropixels probes, we recorded from multi-regional loops connected to the anterior lateral motor cortex (ALM), a circuit node mediating memory-guided directional licking. Neurons encoding sensory stimuli, choices, and actions were distributed across the brain. However, choice coding was concentrated in the ALM and subcortical areas receiving input from the ALM in an ALM-dependent manner. Diverse orofacial movements were encoded in the hindbrain; midbrain; and, to a lesser extent, forebrain. Choice signals were first detected in the ALM and the midbrain, followed by the thalamus and other brain areas. At movement initiation, choice-selective activity collapsed across the brain, followed by new activity patterns driving specific actions. Our experiments provide the foundation for neural circuit models of decision-making and movement initiation.

Although the dynamic instability of microtubules (MTs) is fundamental to many cellular functions, quiescent MTs with unattached free distal ends are commonly present and play important roles in various events to power cellular dynamics. However, how these free MT tips are stabilized remains poorly understood. Here, we report that centrosome and spindle pole protein 1 (CSPP1) caps and stabilizes both plus and minus ends of static MTs. Real-time imaging of laser-ablated MTs in live cells showed deposition of CSPP1 at the newly generated MT ends, whose dynamic instability was concomitantly suppressed. Consistently, MT ends in CSPP1-overexpressing cells were hyper-stabilized, while those in CSPP1-depleted cells were much more dynamic. This CSPP1-elicited stabilization of MTs was demonstrated to be achieved by suppressing intrinsic MT catastrophe and restricting the polymerization. Importantly, CSPP1-bound MTs were resistant to MCAK-mediated depolymerization. These findings delineate a previously uncharacterized CSPP1 activity that integrates MT end capping to orchestrate quiescent MTs.

Expression of the immediate early gene cFos modifies the epigenetic landscape of activated neurons with downstream effects on synaptic plasticity. The production of cFos is inhibited by a long-lived isoform of another Fos family gene, ΔFosB. It has been speculated that this negative feedback mechanism may be critical for protecting episodic memories from being overwritten by new information. Here, we investigate the influence of ΔFosB inhibition on cFos expression and memory. Hippocampal neurons in slice culture produce more cFos on the first day of stimulation compared to identical stimulation on the following day. This downregulation affects all hippocampal subfields and requires histone deacetylation. Overexpression of ΔFosB in individual pyramidal neurons effectively suppresses cFos, indicating that accumulation of ΔFosB is the causal mechanism. Water maze training of mice over several days leads to accumulation of ΔFosB in granule cells of the dentate gyrus, but not in CA3 and CA1. Because the dentate gyrus is thought to support pattern separation and cognitive flexibility, we hypothesized that inhibiting the expression of ΔFosB would affect reversal learning, i.e., the ability to successively learn new platform locations in the water maze. The results indicate that pharmacological HDAC inhibition, which prevents cFos repression, impairs reversal learning, while learning and memory of the initial platform location remain unaffected. Our study supports the hypothesis that epigenetic mechanisms tightly regulate cFos expression in individual granule cells to orchestrate the formation of time-stamped memories.

Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC- FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.

Most mammalian cells prevent viral infection and proliferation by expressing various restriction factors and sensors that activate the immune system. While anti-human immunodeficiency virus type 1 (HIV-1) host restriction factors have been identified, most of them are antagonized by viral proteins. This has severely hindered their development in anti-HIV-1 therapy. Here, we describe CCHC-type zinc-finger-containing protein 3 (ZCCHC3) as a novel anti-HIV-1 factor that is not antagonized by viral proteins. ZCCHC3 suppresses production of HIV-1 and other retroviruses. We show that ZCCHC3 acts by binding to Gag nucleocapsid protein via zinc-finger motifs. This prevents interaction between the Gag nucleocapsid protein and viral genome and results in production of genome-deficient virions. ZCCHC3 also binds to the long terminal repeat on the viral genome via the middle-folded domain, sequestering the viral genome to P-bodies, which leads to decreased viral replication and production. Such a dual antiviral mechanism is distinct from that of any other known host restriction factors. Therefore, ZCCHC3 is a novel potential target in anti-HIV-1 therapy.

The adaptive dynamics of evolving microbial populations takes place on a complex fitness landscape generated by epistatic interactions. The population generically consists of multiple competing strains, a phenomenon known as clonal interference. Microscopic epistasis and clonal interference are central aspects of evolution in microbes, but their combined effects on the functional form of the population’s mean fitness are poorly understood. Here, we develop a computational method that resolves the full microscopic complexity of an evolving population subject to a standard serial dilution protocol. We find that stronger microscopic epistasis gives rise to fitness trajectories with slower growth independent of the number of competing strains, which we quantify with power-law fits and understand mechanistically via a random walk model that neglects dynamical correlations between genes. We show that clonal interference leads to fitness trajectories with faster growth (in functional form) without microscopic epistasis, but has a negligible effect when epistasis is sufficiently strong, indicating that the role of clonal interference depends intimately on the underlying fitness landscape.

The nervous system evolved to enable navigation throughout the environment in the pursuit of resources. Evolutionarily newer structures allowed increasingly complex adaptations but necessarily added redundancy. A dominant view of movement neuroscientists is that there is a one-to-one mapping between brain region and function. However, recent experimental data is hard to reconcile with the most conservative interpretation of this framework, suggesting a degree of functional redundancy during the performance of well-learned, constrained behaviors. This apparent redundancy likely stems from the bidirectional interactions between the various cortical and subcortical structures involved in motor control. We posit that these bidirectional connections enable flexible interactions across structures that change depending upon behavioral demands, such as during acquisition, execution or adaptation of a skill. Observing the system across both multiple actions and behavioral timescales can help isolate the functional contributions of individual structures, leading to an integrated understanding of the neural control of movement.