Biology subjects

Walker, M.

Publications and source records attributed to Walker, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2020

Estimation of sinking velocities using free-falling dynamically scaled models: foraminifera as a test case

The velocity of settling particles is an important determinant of distribution in extinct and extant species with passive dispersal mechanisms, such as plants, corals, and phytoplankton. Here we adapt dynamic scaling, borrowed from engineering, to determine settling velocities. Dynamic scaling leverages physical models with relevant dimensionless numbers matched to achieve similar dynamics to the original object. Previous studies have used flumes, wind tunnels, or towed models to examine fluid flows around objects with known velocities. Our novel application uses free-falling models to determine the unknown sinking velocities of planktonic foraminifera - organisms important to our understanding of the Earths current and historic climate. Using enlarged 3D printed models of microscopic foraminifera tests, sunk in viscous mineral oil to match their Reynolds numbers and drag coefficients, we predict sinking velocities of real tests in seawater. This method can be applied to study other settling particles such as plankton, spores, or seeds. Summary StatementWe developed a novel method to determine the sinking velocities of biologically important microscale particles using 3D printed scale models.

biophysics↗

bioRxiv2020

Pex11 β knockdown decreases peroxisome abundance and reverses the inhibitory effect of palmitate on pancreatic beta-cell function.

AimsReactive oxygen species generated by the peroxisomes and mitochondria contribute to lipotoxicity in pancreatic beta-cells. Through targeted Pex11{beta} knockdown and peroxisome depletion, our aim was to investigate the specific contribution of peroxisomes to palmitate mediated pancreatic beta-cell dysfunction. MethodsMIN6 cells were transfected with probes targeted against Pex11{beta}, a regulator of peroxisome abundance, or with scrambled control probes. Peroxisome abundance was measured by PMP-70 protein expression. 48hrs post transfection, cells were incubated with or without 250M palmitate for a further 48hrs before measurement of reactive oxygen species, mitochondrial respiratory function, and glucose stimulated insulin secretion. ResultsPex11{beta} knockdown decreased target gene expression by more than 80% compared with the scrambled control (P<0.001), leading to decreased PMP-70 expression (p<0.01). Pex11{beta} knockdown decreased palmitate mediated generation of reactive oxygen species (P<0.001), but with no effect on mitochondrial respiratory function. At 25mM glucose, palmitate treatment decreased insulin secretion in the control cells (2.54{+/-}0.25 vs 7.07{+/-}0.83 [mean{+/-}SEM] ng/hr/g protein; P<0.001), with a similar pattern in the Pex11{beta} knockdown cells. However, in the presence of palmitate, insulin secretion was significantly higher in the Pex11{beta} knockdown versus control cells (4.04{+/-}0.46 vs 2.54{+/-}0.25 ng/hr/g protein; p<0.05). ConclusionPex11{beta} knockdown decreased peroxisome abundance, decreased palmitate mediated ROS generation, and reversed the inhibitory effect of palmitate on insulin secretion. These findings highlight a specific and independent role for peroxisomes in pancreatic beta-cell lipotoxicity.

cell biology↗

bioRxiv2020

Time dependencies in the dynamic mechanical behavior of 3D microtissue cell cultures

Characterizing the time-dependent mechanical properties of cells is not only necessary to determine how they deform, but also to fully understand how external forces trigger biochemical-signaling cascades to govern their behavior. Presently mechanical properties are largely assessed by applying local shear or compressive forces on single cells in isolation grown on non-physiological 2D surfaces. In comparison, we developed the microfabricated vacuum actuated stretcher to measure tensile loading of 3D multicellular microtissue cultures. With this approach, we assessed here the time-dependent stress relaxation and recovery responses of microtissues, and quantified the spatial remodeling that follows step length changes. Unlike previous results, stress relaxation and recovery in microtissues measured over a range of step amplitudes and pharmacological treatments followed a stretched exponential behavior describing a broad distribution of inter-related timescales. Furthermore, despite a performed compendium of experiments, all responses led to a single linear relationship between the residual elasticity and degree of stress relaxation, suggesting that these mechanical properties are coupled through interactions between structural elements and the association of cells with their matrix. Lastly, although stress relaxation could be quantitatively and spatially linked to recovery, they differed greatly in their dynamics; while stress recovery behaved as a linear process, relaxation time constants changed with an inverse power law with step size. This assessment of microtissues offers insights into how the collective behavior of cells in a 3D collagen matrix generate the dynamic mechanical properties of tissues, which is necessary to understanding how cells deform and sense mechanical forces in the body.

biophysics↗

bioRxiv2020

Mechanical stretch sustains myofibroblast phenotype and function in microtissues through latent TGF-β1 activation

Fibrosis is a leading cause of death in developed countries that is characterized by a progressive deterioration of tissue mechanical behavior. Developing methods to study tissue mechanics and myofibroblast activation may lead to new targets for therapeutic treatments that are urgently needed. Microtissue arrays are a promising approach to conduct relatively high throughput research into fibrosis as they recapitulate key biomechanical aspects of the disease through a relevant 3D extracellular environment. In early work, our group developed a device called the MVAS-force to stretch microtissues while enabling simultaneous assessment of their dynamic mechanical behavior. Here we investigated TGF-{beta}1 induced fibroblast to myofibroblast differentiation in microtissue cultures using our MVAS-force device through assessing -SMA expression, contractility and stiffness. By doing so, we linked cell-level phenotypic changes to functional changes that characterize the clinical manifestation of fibrotic disease. As expected, TGF-{beta}1 treatment promoted a myofibroblastic phenotype and microtissues became stiffer and possessed increased contractility. Furthermore, these changes were partially reversible upon TGF-{beta}1 withdrawal. In contrast, however, long-term cyclic stretching maintained myofibroblast activation. Furthermore stretching had no effect compared static cultures when TGF-{beta}1 receptors were inhibited and stretching promoted myofibroblast differentiation when given latent TGF-{beta}1. Together these results suggest that external mechanical stretch may activate latent TGF-{beta}1 and might be a powerful stimulus for continued myofibroblast activation to progress fibrosis. Further exploration of this pathway with our approach may yield new insights into myofibroblast activation and more effective therapeutic treatments for fibrosis. Insight boxUsing a novel high-throughput approach, we quantified the effects of dynamic mechanical stretching on the phenotype and function of cells in 3D microtissue cultures during myofibroblast activation with TGF-{beta}1 treatment and subsequent withdrawal. Our findings show that mechanical stretch may activate endogenously produced latent TGF-{beta}1 to maintain the presence and activity of myofibroblasts after tissue injury. Importantly, through this feed forward mechanism, mechanical stretch might be a powerful stimulus that directs tissues away from recovery and towards the development of fibrosis.

cell biology↗

bioRxiv2019

Physical activity reduces colorectal cancer risk independent of BMI - A two-sample Mendelian randomisation study

BackgroundEvidence from observational studies suggests a protective role for physical activity (PA) against colorectal cancer (CRC) risk. However, it has yet to be established a causal relationship. We conducted a two-sample Mendelian randomisation (MR) study to examine causality between physical activity and CRC risk.\n\nMethodsWe used common genetic variants associated with self-reported and accelerometer-based physical activity as instrumental variables (IVs) in this MR study. The IVs were derived from the largest available genome-wide association study (GWAS) of physical activity, namely UK Biobank. We analysed the effect of the IVs for physical activity in a large CRC GWAS that included 31 197 cases and 61 770 controls. We applied inverse variance weighted (IVW) method as the main analysis method.\n\nResultsOur results demonstrate a protective effect between accelerometer-based physical activity and CRC risk (the outlier-adjusted ORIVW was 0.92 per one standard deviation (SD) increase of accelerometer-base physical activity [95% CI: 0.87-0.98, P: 0.01]). The effect between self-reported physical activity and CRC risk was not statistically significant but was also supportive of an inverse association (the outlier-adjusted ORIVW was 0.61 per 1 SD increase of moderate-to-vigorous physical activity [95%CI: 0.36-1.06, P: 0.08]).\n\nConclusionsThe findings of this large MR study show for the first time that objectively measured physical activity is causally implicated in reducing CRC risk. The limitations of the study are that it is based on only two genetic instruments and that it has limited power, despite the study size. Nonetheless, at a population level, these findings provide strong reinforcing evidence to support public health policy measures that encourage exercise, even in obese individuals.

genetics↗

bioRxiv2019

Structural and mechanical remodeling of the cytoskeleton studied in 3D microtissues under acute dynamic stretch

When stretched, cells cultured on 2D substrates share a universal softening and fluidization response that arises from poorly understood remodeling of well-conserved cytoskeletal elements. It is known, however, that the structure and distribution of the cytoskeleton is profoundly influenced by the dimensionality of a cells environment. Therefore, in this study we aimed to determine whether cells cultured in a 3D matrix share this softening behavior and to link it to cytoskeletal remodeling. To achieve this, we developed a high-throughput approach to measure the dynamic mechanical properties of cells and allow for sub-cellular imaging within physiologically relevant 3D microtissues. We found that fibroblast, smooth muscle and skeletal muscle microtissues strain softened but did not fluidize, and upon loading cessation, they regained their initial mechanical properties. Furthermore, microtissue prestress decreased with the strain amplitude to maintain a constant mean tension. This adaptation under an auxotonic condition resulted in lengthening. A filamentous actin cytoskeleton was required, and responses were mirrored by changes to actin remodeling rates and visual evidence of stretch-induced actin depolymerization. Our new approach for assessing cell mechanics has linked behaviors seen in 2D cultures to a 3D matrix, and connected remodeling of the cytoskeleton to homeostatic mechanical regulation of tissues.

biophysics↗

bioRxiv2019

Theta power and theta-gamma coupling support spatial memory retrieval

Hippocampal theta oscillations have been implicated in spatial memory function in both rodents and humans. What is less clear is how hippocampal theta interacts with higher frequency oscillations during spatial memory function, and how this relates to subsequent behaviour. Here we asked ten human epilepsy patients undergoing intracranial EEG recording to perform a desk-top virtual reality spatial memory task, and found that increased theta power in two discrete bands ( low 2-5Hz and high 6-9Hz) during cued retrieval was associated with improved task performance. Similarly, increased coupling between low theta phase and gamma amplitude during the same period was associated with improved task performance. These results support a role of theta oscillations and theta-gamma phase-amplitude coupling in human spatial memory function.

neuroscience↗

bioRxiv2019

Two-Step Small Scale Purification of Recombinant Adeno-Associated Viruses

Recombinant adeno-associated viruses (AAVs) are robust and versatile tools for in vivo gene delivery. Natural and designer capsid variations in AAVs allow for targeted gene delivery to specific cell types. Low immunogenicity and lack of pathogenesis also add to the popularity of this virus as an innocuous gene delivery vector for gene therapy. AAVs are routinely used to express recombinases, sensors, detectors, CRISPR-Cas9 components, or to simply overexpress a gene of interest for functional studies. High production demand has given rise to multiple platforms for production and purification of AAVs. However, most platforms rely heavily on large amounts of starting material and multiple purification steps to produce highly purified viral particles. Often, researchers require several small-scale purified AAVs. Here, we describe a simple and efficient technique for purification of recombinant AAVs from small amounts of starting material in a two-step purification method. In this method, AAVs are released into the packaging cell medium using high salt concentration and pelleted by ultracentrifugation to remove soluble impurities. Then, the resuspended pellet is purified using a protein spin-concentrator. The two-step purification consisting of ultracentrifugation and spin-concentration eliminates the need for fraction collection and the time-consuming evaluation of individual fractionated aliquots for titer and purity. In this method, the resulting AAV preparations are comparable in titer and purity to commercially available samples. This simplified process can be used to rapidly generate highly purified AAV particles in small scale, thereby saving resources.

neuroscience↗

bioRxiv2019

Why Do Long Zinc Finger Proteins have Short Motifs? A case study of ZFY and CTCF reveals non-independent recognition of tandem zinc finger proteins.

The human genome contains around 800 C2H2 Zinc Finger Proteins (ZFPs), and many of them are composed of long tandem arrays of zinc fingers. Current motif prediction models assume longer finger arrays correspond to longer DNA-binding motifs and higher specificity. However, recent experimental efforts to identify ZFP binding sites in vivo contradict this assumption, with many having short motifs. Here, we systematically test how multiple zinc fingers contribute to binding for three model ZFPs: Zinc Finger Y (ZFY), CTCF, and ZNF343. Using ZFY, which contains 13 fingers, we quantitatively characterize its binding specificity with several methods, including Affinity-seq, HT-SELEX, Spec-seq and fluorescence anisotropy, and find evidence for dependent recognition where downstream fingers can recognize some extended motifs only in the presence of an intact core site. For the genomic insulator CTCF, additional high-throughput affinity measurements reveal that its upstream specificity profile depends on the strength of the core, violating presumed additivity and positionindependence. Moreover, the effect of different epigenetic modifications within the core site depends on the strength of flanking upstream site, providing new insight into how the previously identified intellectual disability-causing and cancer-related mutant R567W disrupts upstream recognition and deregulates CTCFs methylation sensitivity. Lastly, we used ZNF343 as example to show that a simple iterative motif analysis strategy based on a small set of prefixed cores can reveal the dependent relationship between cores and upstream motifs. These results establish that the current underestimation of ZFPs motif lengths is due to our lack of understanding of intrinsic properties of tandem zinc finger recognition, including irregular motif structure, variable spacing, and dependent recognition between sub-motifs. These results also motivate a need for better recognition models beyond additive, position-weight matrix to predict ZFP specificities, occupancies, and the molecular mechanisms of disease mutations.

genetics↗

bioRxiv2019

An open resource of structural variation for medical and population genetics

Structural variants (SVs) rearrange large segments of the genome and can have profound consequences for evolution and human diseases. As national biobanks, disease association studies, and clinical genetic testing grow increasingly reliant on genome sequencing, population references such as the Genome Aggregation Database (gnomAD) have become integral for interpreting genetic variation. To date, no large-scale reference maps of SVs exist from high-coverage sequencing comparable to those available for point mutations in protein-coding genes. Here, we constructed a reference atlas of SVs across 14,891 genomes from diverse global populations (54% non-European) as a component of gnomAD. We discovered a rich landscape of 433,371 distinct SVs, including 5,295 multi-breakpoint complex SVs across 11 mutational subclasses, and examples of localized chromosome shattering, as in chromothripsis. The average individual harbored 7,439 SVs, which accounted for 25-29% of all rare protein-truncating events per genome. We found strong correlations between constraint against damaging point mutations and rare SVs that both disrupt and duplicate protein-coding sequence, suggesting intolerance to reciprocal dosage alterations for a subset of tightly regulated genes. We also uncovered modest selection against noncoding SVs in cis-regulatory elements, although selection against protein-truncating SVs was stronger than any effect on noncoding SVs. Finally, we benchmarked carrier rates for medically relevant SVs, finding very large ([≥]1Mb) rare SVs in 3.8% of genomes (~1:26 individuals) and clinically reportable incidental SVs in 0.18% of genomes (~1:556 individuals). These data have been integrated directly into the gnomAD browser (https://gnomad.broadinstitute.org) and will have broad utility for population genetics, disease association, and diagnostic screening.

genomics↗

bioRxiv2019

Isolates from colonic spirochaetosis in humans show high genomic divergence and carry potential pathogenic features but are not detected by 16S amplicon sequencing using standard primers for the human microbiota

Colonic spirochaetosis, diagnosed based on the striking appearance in histological sections, still has an obscure clinical relevance and only few bacterial isolates from this condition have been characterized to date. In a randomized, population-based study in Stockholm, Sweden, 745 healthy individuals underwent colonoscopy with biopsy sampling. In these individuals, 17 (2.3 %) had colonic spirochaetosis, which was associated with eosinophilic infiltration and a three-fold increased risk for irritable bowel syndrome (IBS). We aimed to culture the bacteria and perform whole genome sequencing of the isolates from this unique representative population sample. From 14 out of 17 individuals with spirochaetosis we successfully isolated, cultured and performed whole genome sequencing of in total 17 isolates including the Brachyspira aalborgi type strain 513AT. Also, 16S analysis of the mucosa-associated microbiota was performed in the cases and non-spirochaetosis controls. This is the first report of whole genome analysis of clinical isolates from individuals with colonic spirochaetosis. We found one isolate to be of the species Brachyspira pilosicoli and all remaining isolates were of the species Brachyspira aalborgi. Besides displaying extensive genetic heterogeneity, the isolates harboured several mucin-degrading enzymes and other virulence-associated genes that could confer a pathogenic potential in the human colon. We also showed that 16S amplicon sequencing using standard primers for human microbiota studies fail to detect Brachyspira due to primer incompatibility. This failure to detect colonic spirochaetosis should be taken into consideration in project design and interpretation of gastrointestinal tract microbiota in population-based and clinical settings.

microbiology↗

bioRxiv2019

Prospective, brain-wide labeling of neuronal subclasses with enhancer-driven AAVs

The rapid pace of cell type identification by new single-cell analysis methods has not been met with efficient experimental access to the newly discovered types. To enable flexible and efficient access to specific neural populations in the mouse cortex, we collected chromatin accessibility data from individual cells and clustered the single-cell data to identify enhancers specific for cell classes and subclasses. When cloned into adeno-associated viruses (AAVs) and delivered to the brain by retro-orbital injections, these enhancers drive transgene expression in specific cell subclasses in the cortex. We characterize several enhancer viruses in detail to show that they result in labeling of different projection neuron subclasses in mouse cortex, and that one of them can be used to label the homologous projection neuron subclass in human cortical slices. To enable the combinatorial labeling of more than one cell type by enhancer viruses, we developed a three-color Cre-, Flp- and Nigri-recombinase dependent reporter mouse line, Ai213. The delivery of three enhancer viruses driving these recombinases via a single retroorbital injection into a single Ai213 transgenic mouse results in labeling of three different neuronal classes/subclasses in the same brain tissue. This approach combines unprecedented flexibility with specificity for investigation of cell types in the mouse brain and beyond.

neuroscience↗

bioRxiv2018

Discovery of the role of a SLOG superfamily biological conflict systems associated protein IodA (YpsA) in oxidative stress protection and cell division inhibition in Gram-positive bacteria

Bacteria adapt to different environments by regulating cell division and several conditions that modulate cell division have been documented. Understanding how bacteria transduce environmental signals to control cell division is critical to comprehend the global network of cell division regulation. In this article we describe a role for Bacillus subtilis YpsA, an uncharacterized protein of the SLOG superfamily of nucleotide and ligand-binding proteins, in cell division. We observed that YpsA provides protection against oxidative stress as cells lacking ypsA show increased susceptibility to hydrogen peroxide treatment. We found that increased expression of ypsA leads to cell division inhibition due to defective assembly of FtsZ, the tubulin-like essential protein that marks the sites of cell division. We showed that cell division inhibition by YpsA is linked to glucose availability. We generated YpsA mutants that are no longer able to inhibit cell division. Finally, we show that the role of YpsA is possibly conserved in Firmicutes, as overproduction of YpsA in Staphylococcus aureus also impairs cell division. Therefore, we propose ypsA to be renamed as iodA for inhibitor of division.\n\nIMPORTANCEAlthough key players of cell division in bacteria have been largely characterized, the factors that regulate these division proteins are still being discovered and evidence for the presence of yet-to-be discovered factors has been accumulating. How bacteria sense the availability of nutrients and how that information is used to regulate cell division positively or negatively is less well-understood even though some examples exist in the literature. We discovered that a protein of hitherto unknown function belonging to the SLOG superfamily of nucleotide/ligand-binding proteins, YpsA, influences cell division in Bacillus subtilis by integrating metabolic status such as the availability of glucose. We showed that YpsA is important for oxidative stress response in B. subtilis. Furthermore, we provide evidence that cell division inhibition function of YpsA is also conserved in another Firmicute Staphylococcus aureus. This first report on the role of YpsA (IodA) brings us a step closer in understanding the complete tool set that bacteria have at their disposal to regulate cell division precisely to adapt to varying environmental conditions.

microbiology↗

bioRxiv2018

EWSR1 affects PRDM9-dependent histone 3 methylation and provides a link between recombination hotspots and the chromosome axis

Meiotic recombination in most mammals requires recombination hotspot activation through the action of the histone 3 lysine-4 and lysine-36 methyltransferase PRDM9 to ensure successful double-strand break initiation and repair. Here we show that EWSR1, a protein whose role in meiosis was not previously clarified in detail, binds to both PRDM9 and pREC8, a phosphorylated meiosis-specific cohesin, in male meiotic cells. We created a Ewsr1 conditional knockout mouse models to deplete EWSR1 before the onset of meiosis, and found that absence of EWSR1 causes meiotic arrest with decreased histone trimethylation at meiotic hotspots, impaired DNA double-strand break repair, and reduced crossover number. Our results demonstrate that EWSR1 is essential for promoting PRDM9-dependent histone methylation and normal meiotic progress, possibly by facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis. Author SummaryIn most mammals, including humans and mice, genetic recombination initiates when the meiosis-specific protein PRDM9 binds specific DNA sequences, known as hotspots, at the beginning of the extended prophase I of meiosis, and activates them by trimethylating histone 3 at lysine-4 and lysine-36 on nearby nucleosomes. Although this activation of hotspots is believed to occur on the chromatin loops, the subsequent double-strand break formation and repair occur on a proteinaceous structure known as the chromosome axis. We now show that Ewing sarcoma RNA binding protein 1 (EWSR1) is a key player in early recombination events, binding to PRDM9, promoting PRDM9-dependent histone methylation, and facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis through the meiosis-specific cohesion REC8. As a result of these activities, EWSR1 assures sufficient numbers of properly positioned crossovers in each meiosis.

developmental biology↗

bioRxiv2018

Tissue-specific trans regulation of the mouse epigenome

Although a variety of writers, readers, and erasers of epigenetic modifications are known, we have little information about the underlying regulatory systems controlling the establishment and maintenance of the epigenetic landscape, which varies greatly among cell types. Here, we have explored how natural genetic variation impacts the epigenome in mice. Studying levels of H3K4me3, a histone modification at sites such as promoters, enhancers, and recombination hotspots, we found tissue-specific trans-regulation of H3K4me3 levels in four highly diverse cell types: male germ cells, embryonic stem (ES) cells, hepatocytes and cardiomyocytes. To identify the genetic loci involved, we measured H3K4me3 levels in male germ cells in a mapping population of 60 BXD recombinant inbred lines, identifying extensive trans-regulation primarily controlled by six major histone quantitative trait loci (hQTL). These chromatin regulatory loci act dominantly to suppress H3K4me3, which at hotspots reduces the likelihood of subsequent DNA double-strand breaks. QTL locations do not correspond with enzyme known to metabolize chromatin features. Instead their locations match clusters of zinc finger genes, making these possible candidates that explain the dominant suppression of H3K4me3. Collectively, these data describe an extensive, tissue-specific set of chromatin regulatory loci that control functionally related chromatin sites.

genomics↗

bioRxiv2018

Epilepsy gene therapy using non-integrating lentiviral delivery of an engineered potassium channel gene

Refractory focal neocortical epilepsy is a devastating disease for which there is frequently no effective treatment. Gene therapy represents a promising alternative, but treating epilepsy in this way involves irreversible changes to brain tissue, so vector design must be carefully optimized to guarantee safety without compromising efficacy. We set out to develop an epilepsy gene therapy vector optimized for clinical translation. The gene encoding the voltage-gated potassium channel Kv1.1, KCNA1, was codon-optimized for human expression and mutated to accelerate the channels recovery from inactivation. For improved safety, this engineered potassium channel (EKC) gene was packaged into a non-integrating lentiviral vector under the control of a cell type-specific CAMK2A promoter. In a blinded, randomized, placebo-controlled pre-clinical trial, the EKC lentivector robustly reduced seizure frequency in a rat model of focal neocortical epilepsy characterized by discrete spontaneous seizures. This demonstration of efficacy in a clinically relevant setting, combined with the improved safety conferred by cell type-specific expression and integration-deficient delivery, identify EKC gene therapy as ready for clinical translation in the treatment of refractory focal epilepsy.

neuroscience↗

bioRxiv2018

AAK1 inhibits WNT signaling by promoting clathrin-mediated endocytosis of LRP6

{beta}-catenin-dependent WNT signal transduction governs normal development and adult tissue homeostasis. Inappropriate pathway activity mediates a vast array of human diseases, including bone density disorders, neurodegeneration and cancer. Although several WNT-directed therapeutics are in clinical trials, new targets, compounds and strategies are needed. We performed a gain-of-function screen of the human kinome to identify new druggable regulators of {beta}-catenin-dependent transcription. We found that over-expression of the AP2 Associated Kinase 1 (AAK1) strongly inhibited WNT signaling. Reciprocally, silencing of AAK1 expression or pharmacological inhibition of AAK1 kinase activity using a new, selective and potent small molecule inhibitor activated WNT signaling. This small molecule is a cell active dual AAK1/BMP2K inhibitor that represents the best available tool to study AAK1-dependent signaling pathways. We report that AAK1 and the WNT co-receptor LRP6 physically co-complex and that AAK1 promotes clathrin-mediated endocytosis of LRP6. Collectively, our data support a WNT-induced negative feedback loop mediated by AAK1-driven, clathrin-mediated endocytosis of LRP6.\n\nSummary StatementA gain-of-function screen of the human kinome revealed AAK1 as a negative regulator of WNT signaling. We show that AAK1 promotes clathrin-mediated endocytosis of LRP6, resulting in downregulation of WNT signaling. We use a new selective and potent AAK1/BMP2K small molecule probe to validate our findings.

cell biology↗

bioRxiv2017

A suite of transgenic driver and reporter mouse lines with enhanced brain cell type targeting and functionality

Modern genetic approaches are powerful in providing access to diverse types of neurons within the mammalian brain and greatly facilitating the study of their function. We here report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically-defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

neuroscience↗