Internalization of large double-membrane intercellular vesicles by a clathrin-dependent endocytic process.

Beyond its well-documented role in vesicle endocytosis, clathrin has also been implicated in the internalization of large particles such as viruses, pathogenic bacteria, and even latex beads. We have discovered an additional clathrin-dependent endocytic process that results in the internalization of large, double-membrane vesicles at lateral membranes of cells that are coupled by gap junctions (GJs). GJ channels bridge apposing cell membranes to mediate the direct transfer of electrical currents and signaling molecules from cell to cell. Here, we report that entire GJ plaques, clusters of GJ channels, can be internalized to form large, double-membrane vesicles previously termed annular gap junctions (AGJs). These internalized AGJ vesicles subdivide into smaller vesicles that are degraded by endo/lysosomal pathways. Mechanistic analyses revealed that clathrin-dependent endocytosis machinery-components, including clathrin itself, the alternative clathrin-adaptor Dab2, dynamin, myosin-VI, and actin are involved in the internalization, inward movement, and degradation of these large, intercellular double-membrane vesicles. These findings contribute to the understanding of clathrin's numerous emerging functions.

Double-membrane gap junction internalization requires the clathrin-mediated endocytic machinery.

Direct cell-cell communication mediated by plasma membrane-spanning gap junction (GJ) channels is vital to all aspects of cellular life. Obviously, GJ intercellular communication (GJIC) requires precise regulation, and it is known that controlled biosynthesis and degradation, and channel opening and closing (gating) are exploited. We discovered that cells internalize GJs in response to various stimuli. Here, we report that GJ internalization is a clathrin-mediated endocytic process that utilizes the vesicle-coat protein clathrin, the adaptor proteins adaptor protein complex 2 and disabled 2, and the GTPase dynamin. To our knowledge, we are first to report that the endocytic clathrin machinery can internalize double-membrane vesicles into cells.

Organization and dynamics of growing microtubule plus ends during early mitosis.

A stable cell line expressing EB1-green fluorescent protein was used to image growing microtubule plus ends at the G(2)/M transition. By late prophase growing ends no longer extend to the cell periphery and were not uniformly distributed around each centrosome. Growing ends were much more abundant in the area surrounding the nuclear envelope, and microtubules growing around the nucleus were 1.5 fold longer than those growing in the opposite direction. The growth of longer ends toward the nucleus did not result from a localized faster growth rate, because this rate was approximately 11 microm/min in all directions from the centrosome. Rather, microtubule ends growing toward the nucleus seemed stabilized by dynein/dynactin associated with the nuclear envelope. Injection of p50 into late prophase cells removed dynein from the nuclear envelope, reduced the density of growing ends near the nuclear envelope and resulted in a uniform distribution of growing ends from each centrosome. We suggest that the cell cycle-dependent binding of dynein/dynactin to the nuclear envelope locally stabilizes growing microtubules. Both dynein and microtubules would then be in a position to participate in nuclear envelope breakdown, as described in recent studies.

Porcine Corneal Ocular Reversibility Assay (PorCORA) predicts ocular damage and recovery for global regulatory agency hazard categories.

In this study, we examined the capacity of the Porcine Corneal Ocular Reversibility Assay (PorCORA) to classify the reversibility of ocular effects for 32 test compounds (20 reversible, 12 irreversible) from various chemical classes. PorCORA predicted 28 of 32 compounds correctly when compared to historical rabbit eye test data. The correlation coefficient for PorCORA versus historical rabbit test data was 0.84, based on the last day of damaged cornea reversal. These results demonstrate a high correlation between corneal irritation recovery time in the PorCORA and the rabbit eye. When compared to historical Modified Maximal Average Score (MMAS) in rabbit eyes, PorCORA yielded a correlation coefficient of 0.80, demonstrating ability to predict MMAS. PorCORA was highly predictive of regulatory agency ocular hazard classification categories, resulting in 91% accuracy for EU R41 and GHS Category 1. PorCORA was also predictive of EPA Category I (88% accuracy). Overall, the accuracy (88-91%), sensitivity (79-86%), specificity (94%), positive predictivity (94%), and negative predictivity (85-89%) for all three regulatory classifications indicate that ocular irritation hazardous effects were well predicted by the PorCORA. This study suggests that PorCORA could help discriminate between EU R36 and R41, GHS Categories 1 and 2, and EPA Categories I and II.

Novel cultured porcine corneal irritancy assay with reversibility endpoint.

Several alternative assays exist to assess ocular irritancy without the use of live animals. However, these assays cannot address ocular injury reversibility. Reversibility is an issue critical to regulatory authorities and manufactures of commercial products, as ocular irritation caused by misuse or accidental exposure to a product may cause irreversible eye damage. Here we report the development and initial characterization of a novel ocular irritation assay that addresses ocular injury reversibility. This assay, the Porcine Corneal Ocular Reversibility Assay (PorCORA), uses an air-interface porcine corneal culture system to sustain ex vivo porcine corneas as a model system. These corneas are maintained in culture for 21 days to determine if cornea injury, once inflicted, will reverse. Corneal injury reversibility is measured using Sodium Fluorescein (NaFl) stain to detect compromised epithelial barrier function. In this study, we examined the effects of five compounds on the cultured corneas: phosphate-buffered saline (PBS), 100% Ethanol (EtOH), 3% Sodium Dodecyl Sulfate (SDS), 1% Benzalkonium Chloride (BAK), and 10% Sodium Hydroxide (NaOH). Overall, the persistence of corneal effects between historical Draize rabbit eye data and PorCORA indicates a correlation coefficient of 0.98 (for the five compounds tested) and a correlation coefficient of 0.97 with the Draize modified maximal average score (MMAS). Finally, both fluorescence confocal microscopy and histopathology evidence demonstrates that the PorCORA and NaFl measurements are indicative of actual cellular and tissue damage. PorCORA shows promise as a potential non-animal replacement assay capable of predicting ocular damage reversibility.

Identification of a novel tubulin-destabilizing protein related to the chaperone cofactor E.

Factors that regulate the microtubule cytoskeleton are critical in determining cell behavior. Here we describe the function of a novel protein that we term E-like based on its sequence similarity to the tubulin-specific chaperone cofactor E. We find that upon overexpression, E-like depolymerizes microtubules by committing tubulin to proteosomal degradation. Our data suggest that this function is direct and is based on the ability of E-like to disrupt the tubulin heterodimer in vitro. Suppression of E-like expression results in an increase in the number of stable microtubules and a tight clustering of endocellular membranes around the microtubule-organizing center, while the properties of dynamic microtubules are unaffected. These observations define E-like as a novel regulator of tubulin stability, and provide a link between tubulin turnover and vesicle transport.

Centrosome maturation: measurement of microtubule nucleation throughout the cell cycle by using GFP-tagged EB1.

Understanding how cells regulate microtubule nucleation during the cell cycle has been limited by the inability to directly observe nucleation from the centrosome. To view nucleation in living cells, we imaged GFP-tagged EB1, a microtubule tip-binding protein, and determined rates of nucleation by counting the number of EB1-GFP comets emerging from the centrosome over time. Nucleation rate increased 4-fold between G(2) and prophase and continued to rise through anaphase and telophase, reaching a maximum of 7 times interphase rates. We tested several models for centrosome maturation, including gamma-tubulin recruitment and increased centrosome size. The centrosomal concentration of gamma-tubulin reached a maximum at metaphase, and centrosome size increased through anaphase, whereas nucleation remained high through telophase, implying the presence of additional regulatory processes. Injection of anti-gamma-tubulin antibodies significantly blocked nucleation during metaphase but was less effective during anaphase, suggesting that a nucleation mechanism independent of gamma-tubulin contributes to centrosome function after metaphase.