Biology subjects

Wells, K. M.

Publications and source records attributed to Wells, K. M..

4 recordsLinked to original sources

bioRxiv2022

Nav1.1 in mammalian sensory neurons is required for normal motor behaviors

AO_SCPLOWBSTRACTC_SCPLOWThe voltage-gated sodium channel (NaV), NaV1.1, is well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we identify a new role for NaV1.1 in mammalian proprioception. RNAscope analysis and in vitro patch clamp recordings in genetically identified mouse proprioceptors show ubiquitously channel expression and significant contributions to intrinsic excitability. Notably, genetic deletion of NaV1.1 in sensory neurons caused profound and visible motor coordination deficits in conditional knockout mice of both sexes, similar to conditional Piezo2-knockout animals, suggesting this channel is a major contributor to sensory proprioceptive transmission. Ex vivo muscle afferent recordings conditional knockout mice found that loss of NaV1.1 leads to inconsistent and unreliable proprioceptor firing characterized by action potential failures during static muscle stretch; conversely, afferent responses to dynamic vibrations were unaffected. This suggests that while a combination of Piezo2 and other NaV isoforms are sufficient to elicit activity in response to transient stimuli, NaV1.1 is required for transmission of receptor potentials generated during sustained muscle stretch. Impressively, recordings from afferents of heterozygous conditional knockout animals were similarly impaired, and heterozygous conditional knockout mice also exhibited motor behavioral deficits. Thus, NaV1.1 haploinsufficiency in sensory neurons impairs both proprioceptor function and motor behaviors. Importantly, human patients harboring NaV1.1 loss-of-function mutations often present with motor delays and ataxia; therefore, our data suggest sensory neuron dysfunction contributes to the clinical manifestations of neurological disorders in which NaV1.1 function is compromised. Collectively, we present the first evidence that NaV1.1 is essential for mammalian proprioceptive signaling and behaviors.

neuroscience↗

bioRxiv2021

Diverse pathogens activate the host RIDD pathway to subvert BLOS1-directed immune defense

The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1-dependent decay (RIDD) of mRNAs encoding BLOS1, a protein that promotes endosome-lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1 were resistant to infection. Non-functional Blos1 struggled to assemble the BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Blos1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal-destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus MHV also subverted the RIDD-BLOS1 axis to promote intracellular replication. Our work therefore establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.

microbiology↗

bioRxiv2021

Neurotrophic control of size regulation during axolotl limb regeneration

The mechanisms that regulate the sizing of the regenerating limb in tetrapods such as the Mexican axolotl are unknown. Upon the completion of the developmental stages of regeneration, when the regenerative organ known as the blastema completes patterning and differentiation, the limb regenerate is proportionally small in size. It then undergoes a phase of regeneration that we have called the "tiny-limb" stage, that is defined by rapid growth until the regenerate reaches the proportionally appropriate size. In the current study we have characterized this growth and have found that signaling from the limb nerves is required for its maintenance. Using the regenerative assay known as the Accessory Limb Model, we have found that the size of the limb can be positively and negatively manipulated by nerve abundance. We have additionally developed a new regenerative assay called the Neural Modified-ALM (NM-ALM), which decouples the source of the nerve from the regenerating host environment. Using the NM-ALM we discovered that non-neural extrinsic factors from differently sized host animals do not play a prominent role in determining the size of the regenerating limb. We have also discovered that the regulation of limb size is not autonomously regulated by the limb nerves. Together, these observations show that the limb nerves provide essential and instructive cues to regulate the final size of the regenerating limb.

developmental biology↗

bioRxiv2021

A constitutively expressed fluorescence ubiquitin cell cycle indicator (FUCCI) in axolotls for studying tissue regeneration

Regulation of cell cycle progression is essential for cell proliferation during regeneration following injury. After appendage amputation, the axolotl (Ambystoma mexicanum) regenerates missing structures through an accumulation of proliferating cells known as the blastema. To study cell division during blastema growth, we generated a transgenic line of axolotls that ubiquitously expresses a bicistronic version of the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI). We demonstrate near-ubiquitous expression of FUCCI expression in developing and adult tissues and validate these expression patterns with DNA synthesis and mitosis phase markers. We demonstrate the utility of FUCCI for live and whole-mount imaging, showing the predominantly local contribution of cells during limb and tail regeneration. We also show that spinal cord amputation results in increased proliferation at least 5 mm from the injury. Finally, we use multimodal staining to provide cell type information for cycling cells by combining fluorescence in-situ hybridization, EdU click-chemistry, and immunohistochemistry on a single FUCCI tissue section. This new line of animals will be useful for studying cell cycle dynamics using in-situ endpoint assays and in-vivo imaging in developing and regenerating animals. Summary statementWe generated a ubiquitous transgenic fluorescence ubiquitin cell cycle indicator (FUCCI) axolotl line for examination of cell cycle dynamics during tissue regeneration.

developmental biology↗