No abstract

Genome manipulation methods in C. elegans require microinjecting DNA or ribonucleoprotein complexes into the microscopic core of the gonadal syncytium. These microinjections are technically demanding and represent a key bottleneck for all genome engineering and transgenic approaches in C. elegans. While there have been steady improvements in the ease and efficiency of genetic methods for C. elegans genome manipulation, there have not been comparable advances in the physical process of microinjection. Here, we report a simple and inexpensive method for handling worms using a paintbrush during the injection process that nearly tripled average microinjection rates compared to traditional worm handling methods. We found that the paintbrush increased injection throughput by substantially increasing both injection speeds and post-injection survival rates. In addition to dramatically and universally increasing injection efficiency for experienced personnel, the paintbrush method also significantly improved the abilities of novice investigators to perform key steps in the microinjection process. We expect that this method will benefit the C. elegans community by increasing the speed at which new strains can be generated and will also make microinjection-based approaches less challenging and more accessible to personnel and labs without extensive experience.

Legionella pneumophila is an opportunistic intracellular pathogen responsible for Legionnaires’ disease in humans. Upon infection, L. pneumophila forms the Legionella‐containing vacuole (LCV), shielding itself from the host immune response. From the LCV, L. pneumophila uses a specialized secretion system to translocate over 350 effector proteins into the host cell. Effectors target and manipulate host pathways in order to optimize the host’s cellular environment to support L. pneumophila replication (1). One important cellular process targeted during infection is endocytic recycling. Recycling pathways are generally categorized as slow or fast, depending on the time it takes for endocytosed cargo to return to the plasma membrane. Rab11 is a key regulator of the slow endocytic recycling pathway (2) and it moves along microtubules partly through its interaction with Rab11‐FIP1A. It was previously shown that mutation of Rab11‐FIP1A disrupts transferrin recycling (3). In the context of infection by L. pneumophila, our group has shown that transferrin recycling in human macrophages is inhibited early during infection. We hypothesize that the L. pneumophila effectors protein family SidE is at least partly responsible for this defect. Pull‐down assays revealed that interaction between Rab11 and Rab11‐FIP1A is disrupted in the presence of SdeC. In transiently transfected CHO cells, GFP‐Rab11‐FIP1A is found on vesicles in the perinuclear region. However, when GFP‐Rab11‐FIP1A and mCherry‐SdeC are co‐expressed in CHO cells, we observed that GFP‐Rab11‐FIP1A changed its localization to several enlarged structures. We are investigating whether SdeC’s ubiquitination activity is responsible for disrupting interaction of Rab11 with Rab11‐FIP1A. These results will help establish the effects of ubiquitination by SidE effector proteins on the slow recycling pathway. Support or Funding Information Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectorsE. C.SoC.MattheisE.W.TateG.FrankelG. N.SchroederCanadian Journal of Microbiology619617635Orchestration of cell surface proteins by Rab11T.WelzJ.Wellbourne-WoodE.KerkhoffCell Press247Rab11-FIP1A regulates early trafficking into the recycling endosomesJ. C.ShaferR. E.McRaeE. H.ManningL. A.LapierreJ. R.GoldenringExperimental Cell Research.3402259273

Wnt signaling performs critical functions in development, homeostasis, and disease states. Wnt ligands are secreted signaling proteins that often move between cells to activate signaling across a range of distances and concentrations. In different animals and developmental contexts, Wnts utilize distinct mechanisms for intercellular transport including diffusion, cytonemes and exosomes [1]. Mechanisms for intercellular Wnt dispersal remain controversial in part due to technical challenges with visualizing endogenous Wnt proteins in vivo, which has limited our understanding of Wnt transport dynamics. As a result, the cell-biological bases for long-range Wnt dispersal remain unknown in most instances, and the extent to which differences in Wnt transport mechanisms vary by cell type, organism, and/or ligand remain uncertain. To investigate processes underlying long-range Wnt transport in vivo, we utilized C. elegans as an experimentally tractable model where it is possible to tag endogenous Wnts with fluorescent proteins without disrupting signaling [2]. Live imaging of two endogenously tagged Wnt homologs revealed a novel mode for long-distance Wnt movement in axon-like structures that may complement Wnt gradients generated by diffusion and highlighted cell type-specific Wnt transport processes in vivo.

Signal transduction pathways are heavily dependent on interactions with the cellular environment. The same environmental elements that make food tasty (salt, fat, acid, heat) have profound impacts on many other aspects of protein function. Transducing sensory information involves a change in receptor confirmation on a sensory neuron/end organ (e.g. taste bud), which allows perception of salt, fat, acid and heat. Beyond our perception, however, are the wide‐ranging effects that these environmental elements have on the intracellular signal transduction pathways which impacts every aspect of cell, tissue, organ and organism function. The proteins constituting signalling pathways are constantly receiving environmental information including ionic charges (salt), hydrophobicity (fat), protonation state (acid) and thermal energy (heat). Key Concepts Signal transduction pathways can be altered by environmental fluctuations. Salts dissociate into their separate ionic forms. Regulating ion type and concentration are important for maintaining protein structure, enzyme activity, protein interactions and controlling ion channels. Lipid modifications can influence a protein's microdomain localisation to its target cell and impact ease of intracellular or extracellular diffusion. Fluctuations in pH alter the protonation state of proteins and ion channels which can impact its specificity, affinity to binding partners, structural dynamics and the pathways that are activated. Temperature changes (e.g. extreme hot or cold) can inactivate and denature proteins generally but specifically may gate temperature‐sensitive ion channels [i.e. transient receptor potential ( TRP ) channels]. Changes in temperature can also reveal gain or loss of function phenotypes for mutations that confer less severe phenotypes at physiological temperatures, as exemplified by numerous temperature‐sensitive alleles used in genetics and cell biology experiments.