CUT&Tag for efficient epigenomic profiling of small samples and single cells, bioRxiv, 2019-03-07

AbstractMany chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.

biorxiv genomics 200-500-users 2019

About samples, giving examples Optimized Single Molecule Localization Microscopy, bioRxiv, 2019-03-05

AbstractSuper-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.

biorxiv cell-biology 200-500-users 2019

Machine learning-guided channelrhodopsin engineering enables minimally-invasive optogenetics, bioRxiv, 2019-03-04

We have engineered light-gated channelrhodopsins (ChRs) whose current strength and light sensitivity enable minimally-invasive neuronal circuit interrogation. Current ChR tools applied to the mammalian brain require intracranial surgery for transgene delivery and implantation of invasive fiber-optic cables to produce light-dependent activation of a small volume of brain tissue [~1 mm3]. To enable optogenetics for large brain volumes and without the need for invasive implants, our ChR engineering approach leverages the significant literature of ChR variants to train statistical models for the design of new, high-performance ChRs. With Gaussian Process models trained on a limited experimental set of 102 functionally characterized ChR variants, we designed high-photocurrent ChRs with unprecedented light sensitivity; three of these, ChRger1, ChRger2, and ChRger3, enable optogenetic activation of the nervous system via minimally-invasive systemic transgene delivery with rAAV-PHP.eB, which was not possible previously due to low per-cell transgene copy produced by systemic delivery. These engineered ChRs enable light-induced neuronal excitation without invasive intracranial surgery for virus delivery or fiber optic implantation, i.e. they enable minimally-invasive optogenetics.

biorxiv bioengineering 200-500-users 2019

A unicellular relative of animals generates an epithelium-like cell layer by actomyosin-dependent cellularization, bioRxiv, 2019-03-01

In animals, cellularization of a coenocyte is a specialized form of cytokinesis that results in the formation of a polarized epithelium during early embryonic development. It is characterized by coordinated assembly of an actomyosin network, which drives inward membrane invaginations. However, whether coordinated cellularization driven by membrane invagination exists outside animals is not known. To that end, we investigate cellularization in the ichthyosporean Sphaeroforma arctica, a close unicellular relative of animals. We show that the process of cellularization involves coordinated inward plasma membrane invaginations dependent on an actomyosin network, and reveal the temporal order of its assembly. This leads to the formation of a polarized layer of cells resembling an epithelium. We show that this epithelium-like stage is associated with tightly regulated transcriptional activation of genes involved in cell adhesion. Hereby we demonstrate the presence of a self-organized, clonally-generated, polarized layer of cells in a unicellular relative of animals.

biorxiv evolutionary-biology 200-500-users 2019

Feeding Kinematics And Morphology Of The Alligator Gar (Atractosteus Spatula, Lacépède, 1803) Feeding Mechanics Of Atractosteus Spatula, bioRxiv, 2019-02-28

ABSTRACTModern (lepisosteid) gars are a small clade of seven species and two genera that occupy an important position on the actinopterygian phylogenetic tree as members of the Holostei (Amia + gars), sister-group of the teleost radiation. Often referred to as “living fossils,” these taxa preserve many plesiomorphic characteristics used to interpret and reconstruct early osteichthyan feeding conditions. Less attention, however, has been paid to the functional implications of gar-specific morphology, thought to be related to an exclusively ram-based, lateral-snapping mode of prey capture. Previous studies of feeding kinematics in gars have focused solely on members of the narrow-snouted Lepisosteus genus, and here we expand that dataset to include a member of the broad-snouted sister-genus and largest species of gar, the alligator gar (Atractosteus spatula, Lacépède, 1803). High-speed videography reveals that the feeding system of alligator gars is capable of rapid expansion from anterior-to-posterior, precisely timed in a way that appears to counteract the effects of a bow-wave during ram-feeding and generate a unidirectional flow of water through the feeding system. Reconstructed cranial anatomy based on contrast-enhanced micro-CT data show that a lateral-sliding palatoquadrate, flexible intrasuspensorial joint, pivoting interhyal, and retractable pectoral girdle are all responsible for increasing the range of motion and expansive capabilities of the gar cranial linkage system. Muscular reconstructions and manipulation experiments show that, while the sternohyoideus is the primary input to the feeding system (similar to other “basal” actinopterygians), additional input from the hyoid constrictors and hypaxials play an important role in decoupling and modulating between the dual roles of the sternohyoideus hyoid retraction (jaw opening) and hyoid rotation (pharyngeal expansion) respectively. The data presented here demonstrate an intricate feeding mechanism, capable of precise control with plesiomorphic muscles, that represents one of the many ways the ancestral osteichthyan feeding mechanism has been modified for prey capture.RESEARCH HIGHLIGHTSAlligator gars use a surprisingly expansive cranial linkage system for prey capture that relies on specialized joints for increased mobility and is capable of precise modulation from anterior to posterior using plesiomorphic osteichthyan musculature.

biorxiv evolutionary-biology 200-500-users 2019

Feeding kinematics and morphology of the alligator gar (Atractosteus spatula, Lacépède, 1803), bioRxiv, 2019-02-28

Modern (lepisosteid) gars are a small clade of seven species and two genera that occupy an important position on the actinopterygian phylogenetic tree as members of the Holostei (Amia+ gars), sister-group of the teleost radiation. Often referred to as living fossils, these taxa preserve many plesiomorphic characteristics used to interpret and reconstruct early osteichthyan feeding conditions. Less attention, however, has been paid to the functional implications of gar-specific morphology, thought to be related to an exclusively ram-based, lateral-snapping mode of prey capture. Previous studies of feeding kinematics in gars have focused solely on members of the narrow-snouted Lepisosteus genus, and here we expand that dataset to include a member of the broad-snouted sister-genus and largest species of gar, the alligator gar (Atractosteus spatula, Lacépède, 1803). High-speed videography reveals that the feeding system of alligator gars is capable of rapid expansion from anterior-to-posterior, precisely timed in a way that appears to counteract the effects of a bow-wave during ram-feeding and generate a unidirectional flow of water through the feeding system. Reconstructed cranial anatomy based on contrast-enhanced micro-CT data show that a lateral-sliding palatoquadrate, flexible intrasuspensorial joint, pivoting interhyal, and retractable pectoral girdle are all responsible for increasing the range of motion and expansive capabilities of the gar cranial linkage system. Muscular reconstructions and manipulation experiments show that, while the sternohyoideus is the primary input to the feeding system (similar to other basal actinopterygians), additional input from the hyoid constrictors and hypaxials play an important role in decoupling and modulating between the dual roles of the sternohyoideus hyoid retraction (jaw opening) and hyoid rotation (pharyngeal expansion) respectively. The data presented here demonstrate an intricate feeding mechanism, capable of precise control with plesiomorphic muscles, that represents one of the many ways the ancestral osteichthyan feeding mechanism has been modified for prey capture.

biorxiv evolutionary-biology 200-500-users 2019