In the spindle midzone, microtubules from opposite half-spindles form bundles between segregating chromosomes. Microtubule bundles can either push or restrict chromosome movement during anaphase in different cellular contexts, but how these activities are achieved remains poorly understood. Here, we use high-resolution live-cell imaging to analyze individual microtubule bundles, growing filaments, and chromosome movement in dividing human cells. Within bundles, filament overlap length marked by the cross-linking protein PRC1 decreases during anaphase as chromosome segregation slows. Filament ends within microtubule bundles appear capped despite dynamic PRC1 turnover and submicrometer proximity to growing microtubules. Chromosome segregation distance and rate are increased in two human cell lines when microtubule bundle assembly is prevented via PRC1 knockdown. Upon expressing a mutant PRC1 with reduced microtubule affinity, bundles assemble but chromosome hypersegregation is still observed. We propose that microtubule overlap length reduction, typically linked to pushing forces generated within filament bundles, is needed to properly restrict spindle elongation and position chromosomes within daughter cells.

Multiphoton microscopy is the current method of choice for in vivo deep-tissue imaging. The long laser wavelength suffers less scattering, and the 3D-confined excitation permits the use of scattered signal light. However, the imaging depth is still limited because of the complex refractive index distribution of biological tissue, which scrambles the incident light and destroys the optical focus needed for high resolution imaging. Here, we demonstrate a wavefront-shaping scheme that allows clear imaging through extremely turbid biological tissue, such as the skull, over an extended corrected field of view (FOV). The complex wavefront correction is obtained and directly conjugated to the turbid layer in a noninvasive manner. Using this technique, we demonstrate in vivo submicron-resolution imaging of neural dendrites and microglia dynamics through the intact skulls of adult mice. This is the first observation, to our knowledge, of dynamic morphological changes of microglia through the intact skull, allowing truly noninvasive studies of microglial immune activities free from external perturbations.

Neural damage is a devastating outcome of physical trauma. The glia are one of the main effectors of neuronal repair in the nervous system, but the dynamic interactions between peripheral neurons and Schwann cells during injury and regeneration remain incompletely characterized. Here, we combine laser microsurgery, genetic analysis, high-resolution intravital imaging and lattice light-sheet microscopy to study the interaction between Schwann cells and sensory neurons in a zebrafish model of neurotrauma. We found that chronic denervation by neuronal ablation leads to Schwann-cell death, whereas acute denervation by axonal severing does not affect the overall complexity and architecture of the glia. Neuronal-circuit regeneration begins when Schwann cells extend bridging processes to close the injury gap. Regenerating axons grow faster and directionally after the physiological clearing of distal debris by the Schwann cells. This might facilitate circuit repair by ensuring that axons are guided through unoccupied spaces within bands of Büngner towards their original peripheral target. Accordingly, in the absence of Schwann cells, regenerating axons are misrouted, impairing the re-innervation of sensory organs. Our results indicate that regenerating axons use haptotaxis as a directional cue during the reconstitution of a neural circuit. These findings have implications for therapies aimed at neurorepair, which will benefit from preserving the architecture of the peripheral glia during periods of denervation.

Microelectron diffraction (MicroED) is a new cryo-electron microscopy (cryo-EM) method capable of determining macromolecular structures at atomic resolution from vanishingly small 3D crystals. MicroED promises to solve atomic resolution structures from even the tiniest of crystals, less than a few hundred nanometers thick. MicroED complements frontier advances in crystallography and represents part of the rebirth of cryo-EM that is making macromolecular structure determination more accessible for all. Here we review the concept and practice of MicroED, for both the electron microscopist and crystallographer. Where other reviews have addressed specific details of the technique (Hattne et al., 2015, Shi et al., 2016 and Shi et al., 2013), we aim to provide context and highlight important features that should be considered when performing a MicroED experiment.

Identifying the genes underlying genetically complex traits is of fundamental importance for medicine, agriculture, and evolutionary biology. However, the level of resolution offered by traditional quantitative trait locus (QTL) mapping is usually coarse. We analyze here a trait closely related to fitness, ovariole number. Our initial interspecific mapping between Drosophila sechellia (8 ovarioles/ovary) and D. simulans (15 ovarioles/ovary) identified a major QTL on chromosome 3 and a minor QTL on chromosome 2. To refine the position of the major QTL, we selected 1038 additional recombinants in the region of interest using flanking morphological markers (selective phenotyping). This effort generated approximately one recombination event per gene and increased the mapping resolution by approximately seven times. Our study thus shows that using visible markers to select for recombinants can efficiently increase the resolution of QTL mapping. We resolved the major QTL into two epistatic QTL, QTL3a and QTL3b. QTL3a shows sign epistasis: it has opposite effects in two different genetic backgrounds, the presence vs. the absence of the QTL3b D. sechellia allele. This property of QTL3a allows us to reconstruct the probable order of fixation of the QTL alleles during evolution.

MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.

In vivo direct conversion of differentiated cells holds promise for regenerative medicine; however, improving the conversion efficiency and producing functional target cells remain challenging. Ectopic Atoh1 expression in non-sensory supporting cells (SCs) in mouse cochleae induces their partial conversion to hair cells (HCs) at low efficiency. Here, we performed single-cell RNA sequencing of whole mouse sensory epithelia harvested at multiple time points after conditional overexpression of Atoh1. Pseudotemporal ordering revealed that converted HCs (cHCs) are present along a conversion continuum that correlates with both endogenous and exogenous Atoh1 expression. Bulk sequencing of isolated cell populations and single-cell qPCR confirmed 51 transcription factors, including Isl1, are differentially expressed among cHCs, SCs and HCs. In transgenic mice, co-overexpression of Atoh1 and Isl1 enhanced the HC conversion efficiency. Together, our study shows how high-resolution transcriptional profiling of direct cell conversion can identify co-reprogramming factors required for efficient conversion.

Anemophilous plants described as catapulting pollen explosively into the air have rarely attracted detailed examination. We investigated floral anthesis in a male mulberry tree with high-speed video and a force probe. The stamen was inflexed within the floral bud. Exposure to dry air initially resulted in a gradual movement of the stamen. This caused fine threads to tear at the stomium, ensuring dehiscence of the anther, and subsequently enabled the anther to slip off a restraining pistillode. The sudden release of stored elastic energy in the spring-like filament drove the stamen to straighten in less than 25 μs, and reflex the petals to velocities in excess of half the speed of sound. This is the fastest motion yet observed in biology, and approaches the theoretical physical limits for movements in plants.

Pulsed lasers are key elements in nonlinear bioimaging techniques such as two-photon fluorescence excitation (TPE) microscopy. Typically, however, only a percent or less of the laser power available can be delivered to the sample before photoinduced damage becomes excessive. Here we describe a passive pulse splitter that converts each laser pulse into a fixed number of sub-pulses of equal energy. We applied the splitter to TPE imaging of fixed mouse brain slices labeled with GFP and show that, in different power regimes, the splitter can be used either to increase the signal rate more than 100-fold or to reduce the rate of photobleaching by over fourfold. In living specimens, the gains were even greater: a ninefold reduction in photobleaching during in vivo imaging of Caenorhabditis elegans larvae, and a six- to 20-fold decrease in the rate of photodamage during calcium imaging of rat hippocampal brain slices.

Pulsed lasers are key elements in nonlinear bioimaging techniques such as two-photon fluorescence excitation (TPE) microscopy. Typically, however, only a percent or less of the laser power available can be delivered to the sample before photoinduced damage becomes excessive. Here we describe a passive pulse splitter that converts each laser pulse into a fixed number of sub-pulses of equal energy. We applied the splitter to TPE imaging of fixed mouse brain slices labeled with GFP and show that, in different power regimes, the splitter can be used either to increase the signal rate more than 100-fold or to reduce the rate of photobleaching by over fourfold. In living specimens, the gains were even greater: a ninefold reduction in photobleaching during in vivo imaging of Caenorhabditis elegans larvae, and a six- to 20-fold decrease in the rate of photodamage during calcium imaging of rat hippocampal brain slices.

Commentary: Na Ji came to me early in her postdoc with an idea to reduce photodamage in nonlinear microscopy by splitting the pulses from an ultrafast laser into multiple subpulses of reduced energy. In six weeks, we constructed a prototype pulse splitter and obtained initial results confirming the validity of her vision. Further experiments with Jeff Magee demonstrated that the splitter could be used to increase imaging speed or reduce photodamage in two photon microscopy by one to two orders of magnitude. This project is a great example of how quickly one can react and exploit new ideas in the Janelia environment.