De novo lipid synthesis and polarized prenylation drive cell invasion through basement membrane.

To breach the basement membrane, cells in development and cancer use large, transient, specialized lipid-rich membrane protrusions. Using live imaging, endogenous protein tagging, and cell-specific RNAi during Caenorhabditis elegans anchor cell (AC) invasion, we demonstrate that the lipogenic SREBP transcription factor SBP-1 drives the expression of the fatty acid synthesis enzymes POD-2 and FASN-1 prior to invasion. We show that phospholipid-producing LPIN-1 and sphingomyelin synthase SMS-1, which use fatty acids as substrates, produce lysosome stores that build the AC's invasive protrusion, and that SMS-1 also promotes protrusion localization of the lipid raft partitioning ZMP-1 matrix metalloproteinase. Finally, we discover that HMG-CoA reductase HMGR-1, which generates isoprenoids for prenylation, localizes to the ER and enriches in peroxisomes at the AC invasive front, and that the final transmembrane prenylation enzyme, ICMT-1, localizes to endoplasmic reticulum exit sites that dynamically polarize to deliver prenylated GTPases for protrusion formation. Together, these results reveal a collaboration between lipogenesis and a polarized lipid prenylation system that drives invasive protrusion formation.

Basement membrane dynamics in living animals: Insights and pitfalls.

Recent studies with fluorophore-tagged basement membrane (BM) components have led to remarkable discoveries about BMs but also inconsistent interpretations. Here, we review types of BM dynamics, discuss how we conduct and interpret fluorophore-tagged BM studies, and highlight experimental conditions that are important to consider.

Chronic high-sugar diet in adulthood protects Caenorhabditis elegans from 6-OHDA-induced dopaminergic neurodegeneration.

BACKGROUND: Diets high in saturated fat and sugar, termed "Western diets," have been associated with several negative health outcomes, including increased risk for neurodegenerative disease. Parkinson's disease (PD) is the second most prevalent neurodegenerative disease and is characterized by the progressive death of dopaminergic neurons in the brain. We build upon previous work characterizing the impact of high-sugar diets in Caenorhabditis elegans to mechanistically evaluate the relationship between high-sugar diets and dopaminergic neurodegeneration. RESULTS: Adult high-glucose and high-fructose diets, or exposure from day 1 to 5 of adulthood, led to increased lipid content, shorter lifespan, and decreased reproduction. However, in contrast to previous reports, we found that adult chronic high-glucose and high-fructose diets did not induce dopaminergic neurodegeneration alone and were protective from 6-hydroxydopamine (6-OHDA) induced degeneration. Neither sugar altered baseline electron transport chain function and both increased vulnerability to organism-wide ATP depletion when the electron transport chain was inhibited, arguing against energetic rescue as a basis for neuroprotection. The induction of oxidative stress by 6-OHDA is hypothesized to contribute to its pathology, and high-sugar diets prevented this increase in the soma of the dopaminergic neurons. However, we did not find increased expression of antioxidant enzymes or glutathione levels. Instead, we found evidence suggesting downregulation of the dopamine reuptake transporter dat-1 that could result in decreased 6-OHDA uptake. CONCLUSIONS: Our work uncovers a neuroprotective role for high-sugar diets, despite concomitant decreases in lifespan and reproduction. Our results support the broader finding that ATP depletion alone is insufficient to induce dopaminergic neurodegeneration, whereas increased neuronal oxidative stress may drive degeneration. Finally, our work highlights the importance of evaluating lifestyle by toxicant interactions.

Post-embryonic endogenous expression and localization of LET-60/Ras in C. elegans.

Ras GTPases regulate many developmental and physiological processes and mutations in Ras are associated with numerous human cancers. Here, we report the function, levels, and localization of an N-terminal knock-in of mNeonGreen (mNG) into C. elegans LET-60 /Ras. mNG:: LET-60 interferes with some but not all LET-60 /Ras functions. mNG:: LET-60 is broadly present in tissues, found at different levels in cells, and concentrates in distinct subcellular compartments, including the nucleolus, nucleus, intracellular region, and plasma membrane. These results suggest that mNG:: LET-60 can be a useful tool for determining LET-60 levels and localization once its functionality in a developmental or physiological process is established.

Chronic high-sugar diet in adulthood protects Caenorhabditis elegans from 6-OHDA induced dopaminergic neurodegeneration.

BACKGROUND: Diets high in saturated fat and sugar, termed western diets, have been associated with several negative health outcomes, including increased risk for neurodegenerative disease. Parkinson s Disease (PD) is the second most prevalent neurodegenerative disease and is characterized by the progressive death of dopaminergic neurons in the brain. We build upon previous work characterizing the impact of high sugar diets in Caenorhabditis elegans to mechanistically evaluate the relationship between high sugar diets and dopaminergic neurodegeneration. RESULTS: Non-developmental high glucose and fructose diets led to increased lipid content and shorter lifespan and decreased reproduction. However, in contrast to previous reports, we found that non-developmental chronic high-glucose and high-fructose diets did not induce dopaminergic neurodegeneration alone and were protective from 6-hydroxydopamine (6-OHDA) induced degeneration. Neither sugar altered baseline electron transport chain function, and both increased vulnerability to organism-wide ATP depletion when the electron transport chain was inhibited, arguing against energetic rescue as a basis for neuroprotection. The induction of oxidative stress by 6-OHDA is hypothesized to contribute to its pathology, and high sugar diets prevented this increase in the soma of the dopaminergic neurons. However, we did not find increased expression of antioxidant enzymes or glutathione levels. Instead, we found evidence suggesting alterations to dopamine transmission that could result in decreased 6-OHDA uptake. CONCLUSION: Our work uncovers a neuroprotective role for high sugar diets, despite concomitant decreases in lifespan and reproduction. Our results support the broader finding that ATP depletion alone is insufficient to induce dopaminergic neurodegeneration, whereas increased neuronal oxidative stress may drive degeneration. Finally, our work highlights the importance of evaluating lifestyle by toxicant interactions.

Reciprocal discoidin domain receptor signaling strengthens integrin adhesion to connect adjacent tissues.

Separate tissues connect through adjoining basement membranes to carry out molecular barrier, exchange, and organ support functions. Cell adhesion at these connections must be robust and balanced to withstand independent tissue movement. Yet, how cells achieve synchronized adhesion to connect tissues is unknown. Here, we have investigated this question using the Caenorhabditis elegans utse-seam tissue connection that supports the uterus during egg-laying. Through genetics, quantitative fluorescence, and cell-specific molecular disruption, we show that type IV collagen, which fastens the linkage, also activates the collagen receptor discoidin domain receptor-2 (DDR-2) in both the utse and seam. RNAi depletion, genome editing, and photobleaching experiments revealed that DDR-2 signals through LET-60/Ras to coordinately strengthen an integrin adhesion in the utse and seam that stabilizes their connection. These results uncover a synchronizing mechanism for robust adhesion during tissue connection, where collagen both affixes the linkage and signals to both tissues to bolster their adhesion.

Differential production rates of cytosolic and transmembrane GFP reporters in C. elegans L3 larval uterine cells.

Transgene driven protein expression is an important tool for investigating developmental mechanisms in C. elegans . Here, we have assessed protein production rates and levels in L3 larval uterine cells (UCs). Using ubiquitous promoter driven cytosolic and transmembrane tethered GFP, fluorescence recovery after photobleaching, and quantitative fluorescence analysis, we reveal that cytosolic GFP is produced at an ~two-fold higher rate than transmembrane tethered GFP and accumulates at ~five-fold higher levels in UCs. We also provide evidence that cytosolic GFP in the anchor cell, a specialized UC that mediates uterine-vulval connection, is more rapidly degraded through an autophagy-independent mechanism.

The Caenorhabditis elegans anchor cell transcriptome: ribosome biogenesis drives cell invasion through basement membrane.

Cell invasion through basement membrane (BM) barriers is important in development, immune function and cancer progression. As invasion through BM is often stochastic, capturing gene expression profiles of actively invading cells in vivo remains elusive. Using the stereotyped timing of Caenorhabditis elegans anchor cell (AC) invasion, we generated an AC transcriptome during BM breaching. Through a focused RNAi screen of transcriptionally enriched genes, we identified new invasion regulators, including translationally controlled tumor protein (TCTP). We also discovered gene enrichment of ribosomal proteins. AC-specific RNAi, endogenous ribosome labeling and ribosome biogenesis analysis revealed that a burst of ribosome production occurs shortly after AC specification, which drives the translation of proteins mediating BM removal. Ribosomes also enrich near the AC endoplasmic reticulum (ER) Sec61 translocon and the endomembrane system expands before invasion. We show that AC invasion is sensitive to ER stress, indicating a heightened requirement for translation of ER-trafficked proteins. These studies reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration.

Reciprocal discoidin domain receptor signaling strengthens integrin adhesion to connect adjacent tissues.

Separate tissues connect through adjoining basement membranes to carry out molecular barrier, exchange, and organ support functions. Cell adhesion at these connections must be robust and balanced to withstand independent tissue movement. Yet, how cells achieve synchronized adhesion to connect tissues is unknown. Here, we have investigated this question using the C. elegans utse-seam tissue connection that supports the uterus during egg-laying. Through genetics, quantitative fluorescence, and cell specific molecular disruption, we show that type IV collagen, which fastens the linkage, also activates the collagen receptor discoidin domain receptor 2 (DDR-2) in both the utse and seam. RNAi depletion, genome editing, and photobleaching experiments revealed that DDR-2 signals through LET-60/Ras to coordinately strengthen an integrin adhesion in the utse and seam that stabilizes their connection. These results uncover a synchronizing mechanism for robust adhesion during tissue connection, where collagen both affixes the linkage and signals to both tissues to bolster their adhesion.

The interrater reliability of Modic changes among a potential basivertebral nerve ablation population: Why AC1 may be preferred to kappa.

Interrater reliability of Modic changes is subject to variables which affect consistency in reporting. Given the importance of Modic change identification for basivertebral nerve ablation (BVNA) candidacy, interrater reliability for this specific cohort has not yet been reported. Twenty lumbar magnetic resonance images of potential basivertebral nerve candidates were independently reviewed by two neuroradiologists and two interventional spine physiatrists for the presence and characterization of Modic changes. The kappa value of their agreement on the presence of Modic changes was 0.52 (95% confidence interval [CI] 0.37-0.67), whereas agreement on the type of Modic change was 0.51 (95% CI 0.37-0.65). Using an alternative methodology for measuring interrater reliability (Gwet's AC1) yielded the identification of the presence of Modic changes at AC1 0.51 (95% CI 0.36-0.66), whereas agreement on the type of Modic change was AC1 0.75 (95% CI 0.66-0.83). While less common, AC1 may be preferred in the appropriate cohort to kappa as it mitigates some of the pitfalls to which kappa values may be victim. Ultimately, our results are in-line with previous reports of interrater reliability results for Modic changes in other cohorts and should serve to caution those who perform BVNA regarding interrater agreement of the imaging crux of the procedure.

The Physiatrist's Guide to Cyclist Palsy: A Narrative Review.

TO CLAIM CME CREDITS: Complete the self-assessment activity and evaluation online at http://www.physiatry.org/JournalCME. CME OBJECTIVES: At the conclusion of this educational module, participants will be able to: (1) Describe the possible clinical presentations of Cyclist Palsy based on Ulnar nerve anatomy in the wrist and hand; (2) State the cycling-related risk factors for Cyclist Palsy; and (3) Outline the principles in management for Cyclist Palsy. LEVEL: Advanced. ACCREDITATION: The Association of Academic Physiatrists is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians.The Association of Academic Physiatrists designates this Journal-based CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s) ™. Physicians should only claim credit commensurate with the extent of their participation in the activity.

Hemicentin-mediated type IV collagen assembly strengthens juxtaposed basement membrane linkage.

Basement membrane (BM) matrices surround and separate most tissues. However, through poorly understood mechanisms, BMs of adjacent tissue can also stably link to support organ structure and function. Using endogenous knock-in fluorescent proteins, conditional RNAi, optogenetics, and quantitative live imaging, we identified extracellular matrix proteins mediating a BM linkage (B-LINK) between the uterine utse and epidermal seam cell BMs in Caenorhabditis elegans that supports the uterus during egg-laying. We found that hemicentin is secreted by the utse and promotes fibulin-1 assembly to jointly initiate the B-LINK. During egg-laying, however, both proteins' levels decline and are not required for B-LINK maintenance. Instead, we discovered that hemicentin recruits ADAMTS9/20, which facilitates the assembly of high levels of type IV collagen that sustains the B-LINK during the mechanically active egg-laying period. This work reveals mechanisms underlying BM-BM linkage maturation and identifies a crucial function for hemicentin and fibulin-1 in initiating attachment and type IV collagen in strengthening this specialized form of tissue linkage.

Translational regulation of cell invasion through extracellular matrix-an emerging role for ribosomes.

Many developmental and physiological processes require cells to invade and migrate through extracellular matrix barriers. This specialized cellular behavior is also misregulated in many diseases, such as immune disorders and cancer. Cell invasive activity is driven by pro-invasive transcriptional networks that activate the expression of genes encoding numerous different proteins that expand and regulate the cytoskeleton, endomembrane system, cell adhesion, signaling pathways, and metabolic networks. While detailed mechanistic studies have uncovered crucial insights into pro-invasive transcriptional networks and the distinct cell biological attributes of invasive cells, less is known about how invasive cells modulate mRNA translation to meet the robust, dynamic, and unique protein production needs of cell invasion. In this review we outline known modes of translation regulation promoting cell invasion and focus on recent studies revealing elegant mechanisms that expand ribosome biogenesis within invasive cells to meet the increased protein production requirements to invade and migrate through extracellular matrix barriers.

A light sheet fluorescence microscopy protocol for Caenorhabditis elegans larvae and adults.

Light sheet fluorescence microscopy (LSFM) has become a method of choice for live imaging because of its fast acquisition and reduced photobleaching and phototoxicity. Despite the strengths and growing availability of LSFM systems, no generalized LSFM mounting protocol has been adapted for live imaging of post-embryonic stages of C. elegans. A major challenge has been to develop methods to limit animal movement using a mounting media that matches the refractive index of the optical system. Here, we describe a simple mounting and immobilization protocol using a refractive-index matched UV-curable hydrogel within fluorinated ethylene propylene (FEP) tubes for efficient and reliable imaging of larval and adult C. elegans stages.

A First Estimate of the Annual Prevalence of Basivertebral Nerve Ablation Candidates in a Spine Clinic.

BACKGROUND: Emerging literature supports the use of basivertebral nerve ablation (BVNA) for a specific cohort of patients with chronic low back pain and Type 1 or Type 2 Modic changes from vertebral levels L3-S1. The early literature warrants further evaluation. Studies establishing the efficacy of BVNA use highly selective patient criteria. OBJECTIVE: Provide a first estimate of the prevalence of BVNA candidates in a spine clinic over a year using the foundational studies patient selection criteria? METHODS: A retrospective review of four fellowhsip trained spine physiatrists patient encounters at a large academic medical center using relevant ICD-10 codes to isolate chronic low back pain without radiating symptoms from January 1, 2019 to January 1, 2020. Charts were then reviewed by a team of physicians for exclusionary criteria from the foundational studies which have demonstrated benefit from BVNA. MRI's from qualifying charts which did not meet exclusionary criteria were then independently reviewed by four physician for localization and characterization of Modic changes. RESULTS: The relevant diagnostic codes query yielded 338 unique patient records. Based on exclusionary criteria or lack of imaging availability, 318 charts were eliminated. The remaining 20 charts qualified for imaging review. There were 11 charts in which there was 100% agreement between all reviewers regarding the presence and either Type 1 or Type 2 Modic changes between vertebral levels L3 to S1. Accordingly, the prevalence of eligibility for BVNA was 3% (11/338, 95% CI 1-5%). CONCLUSION: The population which may benefit from BVNA is small. Our study demonstrated that over a year, the prevalence for BVNA candidacy using the foundational studies criteria was 3% (95% CI 1% - 5%). While physicians may be tempted to use less stringent selection criteria in practice, upon doing so they cannot cite the foundational studies as evidence for the outcomes they expect to achieve. Those outcomes will require more studies which formally assess the benefits of BVNA when selection criteria are relaxed.

Visualizing cytoplasmic ATP in C. elegans larvae using PercevalHR.

Measuring ATP levels within the cytosol of living cells in animals is important to understand how cellular activities are energetically supported, but is challenging because of tissue complexity and ATP sensor limitations. In this protocol, we describe how to quantify ATP levels using PercevalHR in C. elegans larvae during anchor cell invasion. PercevalHR is a fluorescent biosensor that reports the cytoplasmic ATP:ADP ratio. The protocol can be adapted to analyze the ATP:ADP ratios within other cell types in C. elegans. For complete details on the use and execution of this protocol, please refer to Garde et al. (2022).

A basement membrane discovery pipeline uncovers network complexity, regulators, and human disease associations.

Basement membranes (BMs) are ubiquitous extracellular matrices whose composition remains elusive, limiting our understanding of BM regulation and function. By developing a bioinformatic and in vivo discovery pipeline, we define a network of 222 human proteins and their animal orthologs localized to BMs. Network analysis and screening in C. elegans and zebrafish uncovered BM regulators, including ADAMTS, ROBO, and TGFß. More than 100 BM network genes associate with human phenotypes, and by screening 63,039 genomes from families with rare disorders, we found loss-of-function variants in LAMA5, MPZL2, and MATN2 and show that they regulate BM composition and function. This cross-disciplinary study establishes the immense complexity of BMs and their impact on in human health.

Localized glucose import, glycolytic processing, and mitochondria generate a focused ATP burst to power basement-membrane invasion.

Invasive cells use transient, energy-consuming protrusions to breach basement membrane (BM) barriers. Using the ATP sensor PercevalHR during anchor cell (AC) invasion in Caenorhabditis elegans, we show that BM invasion is accompanied by an ATP burst from mitochondria at the invasive front. RNAi screening and visualization of a glucose biosensor identified two glucose transporters, FGT-1 and FGT-2, which bathe invasive front mitochondria with glucose and facilitate the ATP burst to form protrusions. FGT-1 localizes at high levels along the invasive membrane, while FGT-2 is adaptive, enriching most strongly during BM breaching and when FGT-1 is absent. Cytosolic glycolytic enzymes that process glucose for mitochondrial ATP production cluster with invasive front mitochondria and promote higher mitochondrial membrane potential and ATP levels. Finally, we show that UNC-6 (netrin), which polarizes invasive protrusions, also orients FGT-1. These studies reveal a robust and integrated energy acquisition, processing, and delivery network that powers BM breaching.

Development of a Cadaver Laboratory Curriculum for Interventional Spine Procedures for Physical Medicine and Rehabilitation Residents.

ABSTRACT: There is no standardized curriculum for teaching interventional spine procedures during residency. The objective of this protocol was to share a curriculum using a cadaver laboratory for teaching Physical Medicine and Rehabilitation residents interventional spine procedures, which can be an effective and safe medium to train residents. This protocol provides a checklist that can guide the residents while they are in the cadaver laboratory with a focus on some of the most common lumbar procedures. Twelve physical medicine and rehabilitation resident's confidence in their ability to maneuver the x-ray image intensifier (C-arm), identify spine anatomy under fluoroscopy, and drive the needle improved after the training curriculum (P < 0.005). Although the cadaver laboratory curriculum is not a replacement for the required Accreditation Council for Graduate Medical Education training, it may serve as a tool to improve resident preparedness for spine procedures.