Actin- and microtubule-based motors contribute to clathrin-independent endocytosis in yeast.

Most eukaryotic cells utilize clathrin-mediated endocytosis as well as multiple clathrin-independent pathways to internalize proteins and membranes. Although clathrin-mediated endocytosis has been studied extensively and many machinery proteins have been identified, clathrin-independent pathways remain poorly characterized by comparison. We previously identified the first known yeast clathrin-independent endocytic pathway, which relies on the actin-modulating GTPase Rho1, the formin Bni1 and unbranched actin filaments, but does not require the clathrin coat or core clathrin machinery proteins. In this study, we sought to better understand clathrin-independent endocytosis in yeast by exploring the role of myosins as actin-based motors, because actin is required for endocytosis in yeast. We find that Myo2, which transports secretory vesicles, organelles and microtubules along actin cables to sites of polarized growth, participates in clathrin-independent endocytosis. Unexpectedly, the ability of Myo2 to transport microtubule plus ends to the cell cortex appears to be required for its role in clathrin-independent endocytosis. In addition, dynein, dynactin, and proteins involved in cortical microtubule capture are also required. Thus, our results suggest that interplay between actin and microtubules contributes to clathrin-independent internalization in yeast.

Racial Differences in Patients Undergoing Anterior Cervical Discectomy and Fusion: A Multi-Site Study.

STUDY DESIGN: This was a retrospective chart review. OBJECTIVE: The objective of this study was to examine disparities within patients undergoing anterior cervical discectomy and fusion (ACDF) at a multi-site tertiary referral center with specific focus on factors related to length of stay (LOS). SUMMARY OF BACKGROUND DATA: There are previously described racial disparities in spinal surgery outcomes and quality metrics. METHODS: A total of 278 consecutive patients undergoing ACDF by 8 different surgeons over a 5-year period were identified retrospectively. Demographic data, including age at time of surgery, sex, smoking status, and self-identified race [White or African American (AA)], as well as surgical data and postoperative course were recorded. Preoperative health status was recorded, and comorbidities were scored by the Charlson Comorbidity Index. Univariable and multivariable linear regression models were employed to quantify the degree to which a patient's LOS was related to their self-identified race, demographics, and perioperative clinical data. RESULTS: Of the 278 patients who received an ACDF, 71.6% (199) self-identified as White and 28.4% (79) identified as AA. AA patients were more likely to have an ACDF due to myelopathy, while White patients were more likely to have an ACDF due to radiculopathy (P=0.001). AA patients had longer LOS by an average of half a day (P=0.001) and experienced a larger percentage of extended stays (P=0.002). AA patients experienced longer overall operation times on average (P=0.001) across all different levels of fusion. AA race was not an independent driver of LOS (ß=0.186; P=0.246). CONCLUSIONS: As hypothesized, and consistent with previous literature on racial surgical disparities, AA race was associated with increased LOS, increased operative times, and increased indication of myelopathy in this study. Additional research is necessary to evaluate the underlying social determinants of health and other factors that may contribute to this study's results. LEVEL OF EVIDENCE: Level III.

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast.

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.