Partially redundant actin genes in Chlamydomonas control flagellum-directed traffic and transition zone organization, bioRxiv, 2017-12-01

ABSTRACTFlagella of the unicellular green alga Chlamydomonas reinhardtii are nearly identical to cilia of mammalian cells and provide an excellent model to study ciliogenesis. These biflagellated cells have two actin genes one encoding a conventional actin (IDA5) and the other encoding a divergent novel actin-like protein (NAP1). Previously, we described a role for actin in the regulation of flagella-building intraflagellar transport machinery. Here, we probe how actin redundancy contributes to this process using a nap1 mutant Chlamydomonas strain. Disruption of a single actin allows normal or slower incorporation but complete flagellar assembly. However, when we disrupt both actins using Latrunculin B (LatB) treatment on the nap1 mutant background, we find flagellar growth from newly synthesized limiting flagellar proteins is actin-dependent. Upon total actin disruption during flagellar assembly, transmission electron microscopy identified an accumulation of Golgi-adjacent vesicles, suggesting impaired vesicular trafficking may be the mechanism by which actin supports flagellar growth from new flagellar proteins. We also find there is a mislocalization of a key transition zone gating and ciliopathy protein, NPHP-4. Extended (2 hour) treatment with LatB, a condition under which NAP1 is upregulated, restores NPHP-4 localization. This suggests NAP1 can perform the functions of conventional actin at the transition zone. Our experiments demonstrate that each stage of flagellar biogenesis requires redundant actin function to varying degrees, with an absolute requirement for these actins in transport of Golgi-adjacent vesicles and flagellar incorporation of newly synthesized proteins.

biorxiv cell-biology 0-100-users 2017

Biomolecular simulations under realistic macroscopic salt conditions, bioRxiv, 2017-11-30

Biomolecular simulations are typically performed in an aqueous environment where the number of ions remains fixed for the duration of the simulation, generally with either a minimally neutralizing ion environment or a number of salt pairs intended to match the macroscopic salt concentration. In contrast, real biomolecules experience local ion environments where the salt concentration is dynamic and may differ from bulk. The degree of salt concentration variability and average deviation from the macroscopic concentration remains, as yet, unknown. Here, we describe the theory and implementation of a Monte Carlo osmostat that can be added to explicit solvent molecular dynamics or Monte Carlo simulations to sample from a semigrand canonical ensemble in which the number of salt pairs fluctuates dynamically during the simulation. The osmostat reproduce the correct equilibrium statistics for a simulation volume that can exchange ions with a large reservoir at a defined macroscopic salt concentration. To achieve useful Monte Carlo acceptance rates, the method makes use of nonequilibrium candidate Monte Carlo (NCMC) moves in which monovalent ions and water molecules are alchemically transmuted using short nonequilibrium trajectories, with a modified Metropolis-Hastings criterion ensuring correct equilibrium statistics for an (Δ𝜇, 𝑁, 𝑝, 𝑇) ensemble. We demonstrate how typical protein (DHFR and the tyrosine kinase Src) and nucleic acid (Drew-Dickerson B-DNA dodecamer) systems exhibit salt concentration distributions that significantly differ from fixed-salt bulk simulations and display fluctuations that are on the same order of magnitude as the average.

biorxiv biophysics 0-100-users 2017

CRISPR-Cas12a target binding unleashes single-stranded DNase activity, bioRxiv, 2017-11-30

AbstractCRISPR-Cas12a (Cpf1) proteins are RNA-guided DNA targeting enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a can be used as a powerful genome editing tool based on its ability to induce genetic changes in cells at sites of double-stranded DNA (dsDNA) cuts. Here we show that RNA-guided DNA binding unleashes robust, non-specific single-stranded DNA (ssDNA) cleavage activity in Cas12a sufficient to completely degrade both linear and circular ssDNA molecules within minutes. This activity, catalyzed by the same active site responsible for site-specific dsDNA cutting, indiscriminately shreds ssDNA with rapid multiple-turnover cleavage kinetics. Activation of ssDNA cutting requires faithful recognition of a DNA target sequence matching the 20-nucleotide guide RNA sequence with specificity sufficient to distinguish between closely related viral serotypes. We find that target-dependent ssDNA degradation, not observed for CRISPR-Cas9 enzymes, is a fundamental property of type V CRISPR-Cas12 proteins, revealing a fascinating parallel with the RNA-triggered general RNase activity of the type VI CRISPR-Cas13 enzymes.One Sentence SummaryCas12a (Cpf1) and related type V CRISPR interference proteins possess non-specific, single-stranded DNase activity upon activation by guide RNA-dependent DNA binding.

biorxiv biochemistry 0-100-users 2017

Estimating the number of missing experiments in a neuroimaging meta-analysis, bioRxiv, 2017-11-28

AbstractCoordinate-based meta-analyses (CBMA) allow researchers to combine the results from multiple fMRI studies with the goal of obtaining results that are more likely to generalise. However, the interpretation of CBMA findings can be impaired by the file drawer problem, a type of publications bias that refers to studies that are carried out but are not published due to lack of significance. Using foci per contrast count data from the BrainMap database, we propose a zero-truncated modelling approach that allows us to estimate the prevalence of non-significant contrasts. We validate our method with simulations and real coordinate data generated from the Human Connectome Project. Application of our method to the data from BrainMap provides evidence for the existence of a file drawer effect, with the rate of missing contrasts estimated as at least 6 per 100 reported.

biorxiv neuroscience 0-100-users 2017

Estimating the prevalence of missing experiments in a neuroimaging meta-analysis, bioRxiv, 2017-11-28

AbstractCoordinate-based meta-analyses (CBMA) allow researchers to combine the results from multiple fMRI experiments with the goal of obtaining results that are more likely to generalise. However, the interpretation of CBMA findings can be impaired by the file drawer problem, a type of publications bias that refers to experiments that are carried out but are not published. Using foci per contrast count data from the BrainMap database, we propose a zero-truncated modelling approach that allows us to estimate the prevalence of non-significant experiments. We validate our method with simulations and real coordinate data generated from the Human Connectome Project. Application of our method to the data from BrainMap provides evidence for the existence of a file drawer effect, with the rate of missing experiments estimated as at least 6 per 100 reported.

biorxiv neuroscience 0-100-users 2017

Prioritized memory access explains planning and hippocampal replay, bioRxiv, 2017-11-28

AbstractTo make decisions, animals must evaluate outcomes of candidate choices by accessing memories of relevant experiences. Yet little is known about which experiences are considered or ignored during deliberation, which ultimately governs choice. Here, we propose a normative theory to predict which memories should be accessed at each moment to optimize future decisions. Using nonlocal “replay” of spatial locations in hippocampus as a window into memory access, we simulate a spatial navigation task where an agent accesses memories of locations sequentially, ordered by utility how much extra reward would be earned due to the computation enabling better choices. This prioritization balances two desiderata the need to evaluate imminent choices, vs. the gain from propagating newly encountered information to predecessor states. We show that this theory offers a unifying account of a range of hitherto disconnected findings in the place cell literature such as the balance of forward and reverse replay, biases in the replayed content, and effects of experience. Accordingly, various types of nonlocal events during behavior and rest are re-interpreted as instances of a single choice evaluation operation, unifying seemingly disparate proposed functions of replay including planning, learning and consolidation, and whose dysfunction may underlie pathologies like rumination and craving.

biorxiv neuroscience 0-100-users 2017