The bacterial injectisome is a syringe-shaped macromolecular nanomachine utilized by many pathogenic Gram-negative bacteria, including the causative agents of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. Bacterial proteins destined for self-assembly and host-cell targeting are translocated by the injectisome in a process known as type III secretion (T3S). The core structure is the ~4 MDa needle complex (NC), built on a foundation of three highly oligomerized ring-forming proteins that create a hollow scaffold spanning the bacterial inner membrane (IM) (24-mer ring-forming proteins PrgH and PrgK in the Salmonella entericaserovar Typhimurium Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS)) and outer membrane (OM) (15-mer InvG, a member of the broadly conserved secretin pore family). An internalized helical needle projects from the NC and bacterium, ultimately forming a continuous passage to the host, for delivery of virulence effectors. Here, we have captured snapshots of the entire prototypical SPI-1 NC in four distinct needle assembly states, including near-atomic resolution, and local reconstructions in the absence and presence of the needle. These structures reveal the precise localization and molecular interactions of the internalized SpaPQR ‘export apparatus’ complex, which is intimately encapsulated and stabilized within the IM rings in the manner of a nanodisc, and to which the PrgJ rod directly binds and functions as an initiator and anchor of needle polymerization. We also describe the molecular details of the extensive and continuous coupling interface between the OM secretin and IM rings, which is remarkably facilitated by a localized 16-mer stoichiometry in the periplasmic-most coupling domain of the otherwise 15-mer InvG oligomer.

Multiple methods have been introduced over the past 30 years to identify the genomic insertion sites of transposable elements and other DNA elements that integrate into genomes. However, each of these methods suffer from limitations that can frustrate attempts to map multiple insertions in a single genome and to map insertions in genomes of high complexity that contain extensive repetitive DNA. I introduce a new method for transposon mapping that is simple to perform, can accurately map multiple insertions per genome, and generates long sequence reads that facilitate mapping to complex genomes. The method, called TagMap, for Tagmentation-based Mapping, relies on a modified Tn5 tagmentation protocol with a single tagmentation adaptor followed by PCR using primers specific to the tranposable element and the adaptor sequence. Several minor modifications to normal tagmentation reagents and protocols allow easy and rapid preparation of TagMap libraries. Short read sequencing starting from the adaptor sequence generates oriented reads that flank and are oriented toward the transposable element insertion site. The convergent orientation of adjacent reads at the insertion site allows straightforward prediction of the precise insertion site(s). A Linux shell script is provided to identify insertion sites from fastq files.

Primary cilia are customized subcellular signaling compartments leveraged to detect signals in diverse physiological contexts. Although prevalent throughout mammalian tissues, primary cilia are not universal. Many non-ciliated cells derive from developmental lineages that include ciliated progenitors; however, little is known about how primary cilia are lost as cells differentiate. Here, we examine how ciliated and non-ciliated states emerge during development and are actively maintained. We highlight several pathways for primary cilia loss, including cilia resorption in pre-mitotic cells, cilia deconstruction in post-mitotic cells, cilia shortening via remodeling, and cilia disassembly preceding multiciliogenesis. Lack of ciliogenesis is known to decrease primary cilia frequency and cause ciliopathies. Failure to maintain cilia can also cause primary cilia to be absent. Conversely, defects in primary cilia suppression or disassembly can lead to the presence of primary cilia in non-ciliated cells. We examine how changes in ciliation states could contribute to tumorigenesis and neurodegeneration.

It is now possible to routinely determine atomic resolution structures by electron cryo-microscopy (cryoEM), facilitated in part by the method known as micro electron-diffraction (MicroED). Since its initial demonstration in 2013, MicroED has helped determine a variety of protein structures ranging in molecular weight from a few hundred Daltons to several hundred thousand Daltons. Some of these structures were novel while others were previously known. The resolutions of structures obtained thus far by MicroED range from 3.2Å to 1.0Å, with most better than 2.5Å. Crystals of various sizes and shapes, with different space group symmetries, and with a range of solvent content have all been studied by MicroED. The wide range of crystals explored to date presents the community with a landscape of opportunity for structure determination from nano crystals. Here we summarize the lessons we have learned during the first few years of MicroED, and from our attempts at the first ab initio structure determined by the method. We re-evaluate theoretical considerations in choosing the appropriate crystals for MicroED and for extracting the most meaning out of measured data. With more laboratories worldwide adopting the technique, we speculate what the first decade might hold for MicroED.

We tested whether transcription activator-like effectors (TALEs) could mediate repression and activation of endogenous enhancers in the Drosophila genome. TALE repressors (TALERs) targeting each of the five even-skipped (eve) stripe enhancers generated repression specifically of the focal stripes. TALE activators (TALEAs) targeting the eve promoter or enhancers caused increased expression primarily in cells normally activated by the promoter or targeted enhancer, respectively. This effect supports the view that repression acts in a dominant fashion on transcriptional activators and that the activity state of an enhancer influences TALE binding or the ability of the VP16 domain to enhance transcription. In these assays, the Hairy repression domain did not exhibit previously described long-range transcriptional repression activity. The phenotypic effects of TALER and TALEA expression in larvae and adults are consistent with the observed modulations of eve expression. TALEs thus provide a novel tool for detection and functional modulation of transcriptional enhancers in their native genomic context.

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.