Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery.

The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early- and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation.

TFG regulates inner COPII coat recruitment to facilitate anterograde secretory protein transport.

Coat protein complex II (COPII) governs the initial steps of biosynthetic secretory protein transport from the endoplasmic reticulum (ER), facilitating the movement of a wide variety of cargoes. Here, we demonstrate that Trk-fused gene (TFG) regulates the rate at which inner COPII coat proteins are concentrated at ER subdomains. Specifically, in cells lacking TFG, the GTPase-activating protein (GAP) Sec23 accumulates more rapidly at budding sites on the ER as compared to control cells, potentially altering the normal timing of GTP hydrolysis on Sar1. Under these conditions, anterograde trafficking of several secretory cargoes is delayed, irrespective of their predicted size. We propose that TFG controls the local, freely available pool of Sec23 during COPII coat formation and limits its capacity to prematurely destabilize COPII complexes on the ER. This function of TFG enables it to act akin to a rheostat, promoting the ordered recruitment of Sec23, which is critical for efficient secretory cargo export.

Nutrient deprivation alters the rate of COPII coat assembly to tune secretory protein transport.

Co-assembly of the multilayered coat protein complex II (COPII) with the Sari GTPase at subdomains of the endoplasmic reticulum (ER) enables secretory cargoes to be concentrated efficiently within nascent transport intermediates, which subsequently deliver their contents to ER-Golgi intermediate compartments. Here, we define the spatiotemporal accumulation of native COPII subunits and secretory cargoes at ER subdomains under differing nutrient availability conditions using a combination of CRISPR/Cas9-mediated genome editing and live cell imaging. Our findings demonstrate that the rate of inner COPII coat assembly serves as a determinant for the pace of cargo export, irrespective of COPII subunit expression levels. Moreover, increasing inner COPII coat assembly kinetics is sufficient to rescue cargo trafficking deficits caused by acute nutrient limitation in a manner dependent on Sar1 GTPase activity. Our findings are consistent with a model in which the rate of inner COPII coat formation acts as an important control point to regulate cargo export from the ER.

Nutrient deprivation alters the rate of COPII subunit recruitment at ER subdomains to tune secretory protein transport.

Co-assembly of the multilayered coat protein complex II (COPII) with the Sar1 GTPase at subdomains of the endoplasmic reticulum (ER) enables secretory cargoes to be concentrated efficiently within nascent transport intermediates, which subsequently deliver their contents to ER-Golgi intermediate compartments. Here, we define the spatiotemporal accumulation of native COPII subunits and secretory cargoes at ER subdomains under differing nutrient availability conditions using a combination of CRISPR/Cas9-mediated genome editing and live cell imaging. Our findings demonstrate that the rate of inner COPII coat recruitment serves as a determinant for the pace of cargo export, irrespective of COPII subunit expression levels. Moreover, increasing inner COPII coat recruitment kinetics is sufficient to rescue cargo trafficking deficits caused by acute nutrient limitation. Our findings are consistent with a model in which the rate of inner COPII coat addition acts as an important control point to regulate cargo export from the ER.

The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

Coat protein complex II (COPII) plays an integral role in the packaging of secretory cargoes within membrane-enclosed transport carriers that leave the endoplasmic reticulum (ER) from discrete subdomains. Lipid bilayer remodeling necessary for this process is driven initially by membrane penetration mediated by the Sar1 GTPase and further stabilized by assembly of a multilayered complex of several COPII proteins. However, the relative contributions of these distinct factors to transport carrier formation and protein trafficking remain unclear. Here, we demonstrate that anterograde cargo transport from the ER continues in the absence of Sar1, although the efficiency of this process is dramatically reduced. Specifically, secretory cargoes are retained nearly five times longer at ER subdomains when Sar1 is depleted, but they ultimately remain capable of being translocated to the perinuclear region of cells. Taken together, our findings highlight alternative mechanisms by which COPII promotes transport carrier biogenesis.

Quantifying Total and Out-of-Pocket Costs Associated With Head and Neck Cancer Survivorship.

Importance: Oncologic treatment is costly to the health care system and to individuals, but patients with head and neck cancer (HNC) also have long-term care needs after treatment. Survivors of HNC require specific consideration given their rapidly growing numbers. This subpopulation of cancer survivors often experiences long-term treatment-associated morbidity. Objective: To describe the total and out-of-pocket (OOP) costs associated with HNC survivorship and the risk factors for financial toxicity among this population. Design, Setting, and Participants: This was a retrospective review and economic evaluation of a cohort of US adults with a diagnosis of HNC from 2006 to 2018. The study used data the from IBM MarketScan Commercial Claims Database. Data were analyzed from November 2020 to June 2022. Exposures: Treatment for HNC. Main Outcomes and Measures: Total and OOP medical costs were assessed monthly and reported relative to the date of HNC diagnosis. The primary outcome was the difference between a patient's mean monthly survivorship costs (13-60 months after diagnosis) and mean monthly baseline costs (7-12 months before diagnosis). Univariate and multivariable linear regression models were created for total and OOP costs to generate coefficient estimates with 95% CIs. Results: The study cohort of this economic evaluation included 19 098 patients with HNC (median [range] age, 56 [18-64] years; 14 144 [74.1%] men and 4954 [25.9%] women; race and ethnicity were not considered). Throughout the survivorship period, median total and OOP costs were $372 per month and $31 per month higher than baseline costs, respectively, with variation in expenses by demographic information, health plan type, and oncologic variables. In the multivariable model, greater total and OOP excess survivorship costs were associated with female sex ($343/mo; 95% CI, $126 to $560 and $9/mo; 95% CI, $4 to $14). Highest and lowest total excess survivorship costs associated with cancer site were seen for hypopharyngeal ($1908/mo; 95% CI, $1102 to $2714) and oropharyngeal cancers (-$703/mo; 95% CI, -$967 to -$439) vs oral cavity cancers. Compared with surgery or radiation therapy alone, multimodal treatment was generally associated with excess OOP survivorship costs. Conclusions and Relevance: The findings of this retrospective economic evaluation review suggest that the costs of HNC survivorship remain persistently elevated above baseline costs for at least 5 years after diagnosis. High survivorship costs were associated with female sex, hypopharyngeal tumors, and treatment with multimodal therapy. Practitioners should seek to minimize costs for these patients at higher-risk of financial toxicity after treatment and work to provide directed supportive services.

Analysis of academic publishing output among 1634 successful applicants in the 2011-2018 neurosurgery residency match.

BACKGROUND: Research productivity is a key criterion for applicant selection reported by residency program directors. Research volume reported on neurosurgery residency applications has risen steadily over the past decade. OBJECTIVE: Perform retrospective bibliographic searches of successful applicants who matched into U.S. neurosurgery residency programs from 2011 to 2018, and assess the relationship between academic publishing and residency placement. METHODS: Gender, MD/PhD status, U.S. News research ranking of medical school, and international medical graduate status (IMG) were determined for 1634 successful applicants from 2011 to 2018. Indexed publications before and after the start of residency were tabulated by Scopus®. Publication counts were stratified by first author, basic/clinical science, case reports, reviews, or other research. We then compared publishing trends across demographic variables and match cohorts. RESULTS: Average pre-residency publications increased from 2.6 [1.7, 3.4] in 2011 to 6.5 [5.1, 7.9] in 2018. Men, PhD-holders, Top 20 and Top 40 U.S. medical school graduates, and IMGs had higher pre-residency publication counts overall. After stratifying by match cohort, however, there was no significant effect of gender on pre-residency publications. Applicants matching into residency programs with highly ranked affiliated hospitals had significantly higher pre-residency publications. CONCLUSION: Publishing volume of successful neurosurgery applicants in the U.S. has risen recently and is associated with the stature of matched residency programs. Given the gap between verifiable and claimed research on residency applications, attention is needed to objectively evaluate research credentials in the selection process. The impending phase out of USMLE step 1 scores may increase emphasis on academic productivity.

Downshifting Yeast Dominance: Cell Physiology and Phospholipid Composition Are Altered With Establishment of the [GAR +] Prion in Saccharomyces cerevisiae.

Establishment of the [GAR +] prion in Saccharomyces cerevisiae reduces both transcriptional expression of the HXT3 hexose transporter gene and fermentation capacity in high sugar conditions. We evaluated the impact of deletion of the HXT3 gene on the expression of [GAR +] prion phenotype in a vineyard isolate, UCD932, and found that changes in fermentation capacity were observable even with complete loss of the Hxt3 transporter, suggesting other cellular functions affecting fermentation rate may be impacted in [GAR +] strains. In a comparison of isogenic [GAR +] and [gar -] strains, localization of the Pma1 plasma membrane ATPase showed differences in distribution within the membrane. In addition, plasma membrane lipid composition varied between the two cell types. Oxygen uptake was decreased in prion induced cells suggesting membrane changes affect plasma membrane functionality beyond glucose transport. Thus, multiple cell surface properties are altered upon induction of the [GAR +] prion in addition to changes in expression of the HXT3 gene. We propose a model wherein [GAR +] prion establishment within a yeast population is associated with modulation of plasma membrane functionality, fermentation capacity, niche dominance, and cell physiology to facilitate growth and mitigate cytotoxicity under certain environmental conditions. Down-regulation of expression of the HXT3 hexose transporter gene is only one component of a suite of physiological differences. Our data show the [GAR +] prion state is accompanied by multiple changes in the yeast cell surface that prioritize population survivability over maximizing metabolic capacity and enable progeny to establish an alternative adaptive state while maintaining reversibility.