ABSTRACT
In this paper, we use narrative analysis to examine the case study of "Nicole" (pseudonym), a student in a science, technology, engineering, and mathematics (STEM) program who transferred from a 2-year college (TYC) to a 4-year college (FYC). We draw from longitudinal qualitative data that follow Nicole's experience pre- and posttransfer, while acknowledging the impact of her experience at the TYC. We use an anti-deficit lens by highlighting the role of supporting characters for Nicole, especially at the TYC. Narrative analysis of Nicole's experiences highlights differences in her sense of community at the two different institutions. Organizing our data in these narrative components revealed how impactful supporting characters are in Nicole's story and how drastically they can shape the outcome of scenes in her story. Instructors and programmatic staff at FYCs who aim to better support transfer students in their transition can learn from the kinds of scenes Nicole cited as helpful in her time at the TYC as well as the FYC. It is our aim in sharing Nicole's story to provide guidelines for how faculty and program directors could be impactful supporting characters to create welcoming settings for transfer students.
Subject(s)
Engineering , Students , Engineering/education , Female , Humans , Mathematics , Technology/education , UniversitiesABSTRACT
Clathrin-mediated endocytosis (CME) occurs continuously at the plasma membrane of eukaryotic cells. However, when a vesicle forms and what cargo it contains are unpredictable. We recently developed a system to trigger CME on-demand. This means that we can control when endocytosis is triggered and the design means that the cargo that is internalized is predetermined. The method is called hot-wired CME because several steps and proteins are bypassed in our system. In this chapter, we describe in detail how to use the hot-wiring system to trigger endocytosis in human cell lines and how to image the vesicles that form using microscopy and finally, how to analyze those images.
Subject(s)
Clathrin-Coated Vesicles/metabolism , Clathrin/metabolism , Endocytosis/physiology , Molecular Imaging , Clathrin/chemistry , Clathrin-Coated Vesicles/ultrastructure , Fluorescent Antibody Technique , HeLa Cells , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Microscopy, Confocal , Molecular Imaging/methodsABSTRACT
Clathrin-mediated endocytosis (CME) is the major route of receptor internalization at the plasma membrane. Analysis of constitutive CME is difficult because the initiation of endocytic events is unpredictable. When and where a clathrin-coated pit will form and what cargo it will contain are difficult to foresee. Here we describe a series of genetically encoded reporters that allow the initiation of CME on demand. A clathrin-binding protein fragment ("hook") is inducibly attached to an "anchor" protein at the plasma membrane, which triggers the formation of new clathrin-coated vesicles. Our design incorporates temporal and spatial control by the use of chemical and optogenetic methods for inducing hook-anchor attachment. Moreover, the cargo is defined. Because several steps in vesicle creation are bypassed, we term it "hot-wiring." We use hot-wired endocytosis to describe the functional interactions between clathrin and AP2. Two distinct sites on the ß2 subunit, one on the hinge and the other on the appendage, are necessary and sufficient for functional clathrin engagement.
Subject(s)
Adaptor Protein Complex 2/genetics , Clathrin-Coated Vesicles/metabolism , Clathrin/genetics , Coated Pits, Cell-Membrane/metabolism , Endocytosis/genetics , Epithelial Cells/metabolism , Adaptor Protein Complex 2/metabolism , Cell Line , Clathrin/metabolism , Clathrin-Coated Vesicles/ultrastructure , Coated Pits, Cell-Membrane/ultrastructure , Epithelial Cells/ultrastructure , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Metabolic Engineering/methods , Optogenetics , Protein Binding , Protein Subunits/genetics , Protein Subunits/metabolism , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/ultrastructure , Signal Transduction , Tacrolimus/pharmacology , Tacrolimus Binding Proteins/genetics , Tacrolimus Binding Proteins/metabolism , Red Fluorescent ProteinABSTRACT
Endocytosis is the physical battle to form a new vesicle in the face of counteracting forces, such as membrane tension. Skruzny et al. (2015) and Miller et al. (2015) now shed light on endocytic proteins that bear a "Helix 0" and on the proteins' role in the struggle to make clathrin-coated vesicles.
