Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein-DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

The mouse is an increasingly prominent model for the analysis of mammalian neuronal circuits. Neural circuits ultimately have to be probed during behaviors that engage the circuits. Linking circuit dynamics to behavior requires precise control of sensory stimuli and measurement of body movements. Head-fixation has been used for behavioral research, particularly in non-human primates, to facilitate precise stimulus control, behavioral monitoring and neural recording. However, choice-based, perceptual decision tasks by head-fixed mice have only recently been introduced. Training mice relies on motivating mice using water restriction. Here we describe procedures for head-fixation, water restriction and behavioral training for head-fixed mice, with a focus on active, whisker-based tactile behaviors. In these experiments mice had restricted access to water (typically 1 ml/day). After ten days of water restriction, body weight stabilized at approximately 80% of initial weight. At that point mice were trained to discriminate sensory stimuli using operant conditioning. Head-fixed mice reported stimuli by licking in go/no-go tasks and also using a forced choice paradigm using a dual lickport. In some cases mice learned to discriminate sensory stimuli in a few trials within the first behavioral session. Delay epochs lasting a second or more were used to separate sensation (e.g. tactile exploration) and action (i.e. licking). Mice performed a variety of perceptual decision tasks with high performance for hundreds of trials per behavioral session. Up to four months of continuous water restriction showed no adverse health effects. Behavioral performance correlated with the degree of water restriction, supporting the importance of controlling access to water. These behavioral paradigms can be combined with cellular resolution imaging, random access photostimulation, and whole cell recordings.

3D snapshot microscopy enables fast volumetric imaging by capturing a 3D volume in a single 2D camera image and performing computational reconstruction. Fast volumetric imaging has a variety of biological applications such as whole brain imaging of rapid neural activity in larval zebrafish. The optimal microscope design for this optical 3D-to-2D encoding is both sample- and task-dependent, with no general solution known. Deep learning based decoders can be combined with a differentiable simulation of an optical encoder for end-to-end optimization of both the deep learning decoder and optical encoder. This technique has been used to engineer local optical encoders for other problems such as depth estimation, 3D particle localization, and lensless photography. However, 3D snapshot microscopy is known to require a highly non-local optical encoder which existing UNet-based decoders are not able to engineer. We show that a neural network architecture based on global kernel Fourier convolutional neural networks can efficiently decode information from multiple depths in a volume, globally encoded across a 3D snapshot image. We show in simulation that our proposed networks succeed in engineering and reconstructing optical encoders for 3D snapshot microscopy where the existing state-of-the-art UNet architecture fails. We also show that our networks outperform the state-of-the-art learned reconstruction algorithms for a computational photography dataset collected on a prototype lensless camera which also uses a highly non-local optical encoding.

Aquaporin-9, an aquaglyceroporin present in diverse tissues, is unique among aquaporins because it is not only permeable to water, urea and glycerol, but also allows passage of larger uncharged solutes. Single particle analysis of negatively stained recombinant rat aquaporin-9 revealed a particle size characteristic of the tetrameric organization of all members of the aquaporin family. Reconstitution of aquaporin-9 into two-dimensional crystals enabled us to calculate a projection map at 7 A resolution. The projection structure indicates a tetrameric structure, similar to GlpF, with each square-like monomer forming a pore. A comparison of the pore-lining residues between the crystal structure of GlpF and a homology model of aquaporin-9 locates substitutions in these residues predominantly to the hydrophobic edge of the tripathic pore of GlpF, providing first insights into the structural basis for the broader substrate specificity of aquaporin-9.

The brain receives information about the direction of object motion from several types of retinal ganglion cells (RGCs). On-Off direction-selective (DS) RGCs respond preferentially to stimuli moving quickly in one of four directions and provide a significant (but difficult to quantify) fraction of RGC input to the SC. On DS RGCs, in comparison, respond preferentially to stimuli moving slowly in one of three directions and are thought to only target retinorecipient nuclei comprising the accessory optic system, e.g., the medial terminal nucleus (MTN). To determine the fraction of SC-projecting RGCs that exhibit direction selectivity, and the specificity with which On-Off and On DS RGCs target retinorecipient areas, we performed optical and electrophysiological recordings from RGCs retrogradely labeled from the mouse SC and MTN. We found, surprisingly, that both On-Off and On DS RGCs innervate the SC; collectively they constitute nearly 40% of SC-projecting RGCs. In comparison, only On DS RGCs project to the MTN. Subsequent experiments revealed that individual On DS RGCs innervate either the SC or MTN and exhibit robust projection-specific differences in somatodendritic morphology, cellular excitability, and light-evoked activity; several projection-specific differences in the output of On DS RGCs correspond closely to differences in excitatory synaptic input the cells receive. Our results reveal a robust projection of On DS RGCs to the SC, projection-specific differences in the response properties of On DS RGCs, and biophysical and synaptic mechanisms that underlie these functional differences.

During low-frequency firing, action potentials actively invade the dendrites of CA1 pyramidal neurons. At higher firing rates, however, activity-dependent processes result in the attenuation of back-propagating action potentials, and propagation failures occur at some dendritic branch points. We tested two major hypotheses related to this activity-dependent attenuation of back-propagating action potentials: (1) that it is mediated by a prolonged form of sodium channel inactivation and (2) that it is mediated by a persistent dendritic shunt activated by back-propagating action potentials. We found no evidence for a persistent shunt, but we did find that cumulative, prolonged inactivation of sodium channels develops during repetitive action potential firing. This inactivation is significant after a single action potential and continues to develop during several action potentials thereafter, until a steady-state sodium current is established. Recovery from this form of inactivation is much slower than its induction, but recovery can be accelerated by hyperpolarization. The similarity of these properties to the time and voltage dependence of attenuation and recovery of dendritic action potentials suggests that dendritic sodium channel inactivation contributes to the activity dependence of action potential back-propagation in CA1 neurons. Hence, the biophysical properties of dendritic sodium channels will be important determinants of action potential-mediated effects on synaptic integration and plasticity in hippocampal neurons.

Selective transcription of human mitochondrial DNA requires a transcription factor (mtTF) in addition to an essentially nonselective RNA polymerase. Partially purified mtTF is able to sequester promoter-containing DNA in preinitiation complexes in the absence of mitochondrial RNA polymerase, suggesting a DNA-binding mechanism for factor activity. Functional domains, required for positive transcriptional regulation by mtTF, are identified within both major promoters of human mtDNA through transcription of mutant promoter templates in a reconstituted in vitro system. These domains are essentially coextensive with DNA sequences protected from nuclease digestion by mtTF-binding. Comparison of the sequences of the two mtTF-responsive elements reveals significant homology only when one sequence is inverted; the binding sites are in opposite orientations with respect to the predominant direction of transcription. Thus mtTF may function bidirectionally, requiring additional protein-DNA interactions to dictate transcriptional polarity. The mtTF-responsive elements are arrayed as direct repeats, separated by approximately 80 bp within the displacement-loop region of human mitochondrial DNA; this arrangement may reflect duplication of an ancestral bidirectional promoter, giving rise to separate, unidirectional promoters for each strand.

In mammals, cerebellar neurons are categorized as glutamatergic or GABAergic, and are derived from progenitors that express the proneural genes atoh1 or ptf1a, respectively. In zebrafish, three atoh1 genes, atoh1a, atoh1b, and atoh1c, are expressed in overlapping but distinct expression domains in the upper rhombic lip (URL): ptf1a is expressed exclusively in the ventricular zone (VZ). Using transgenic lines expressing fluorescent proteins under the control of the regulatory elements of atoh1a and ptf1a, we traced the lineages of the cerebellar neurons. The atoh1+ progenitors gave rise not only to granule cells but also to neurons of the anteroventral rhombencephalon. The ptf1a+ progenitors generated Purkinje cells. The olig2+ eurydendroid cells, which are glutamatergic, were derived mostly from ptf1a+ progenitors in the VZ but some originated from the atoh1+ progenitors in the URL. In the adult cerebellum, atoh1a, atoh1b, and atoh1c are expressed in the molecular layer of the valvula cerebelli and of the medial corpus cerebelli, and ptf1a was detected in the VZ. The proneural gene expression patterns coincided with the sites of proliferating neuronal progenitors in the adult cerebellum. Our data indicate that proneural gene-linked neurogenesis is evolutionarily conserved in the cerebellum among vertebrates, and that the continuously generated neurons help remodel neural circuits in the adult zebrafish cerebellum.