Mechanism of actin capping protein recruitment and turnover during clathrin-mediated endocytosis.

Clathrin-mediated endocytosis depends on polymerization of a branched actin network to provide force for membrane invagination. A key regulator in branched actin network formation is actin capping protein (CP), which binds to the barbed end of actin filaments to prevent the addition or loss of actin subunits. CP was thought to stochastically bind actin filaments, but recent evidence shows CP is regulated by a group of proteins containing CP-interacting (CPI) motifs. Importantly, how CPI motif proteins function together to regulate CP is poorly understood. Here, we show Aim21 and Bsp1 work synergistically to recruit CP to the endocytic actin network in budding yeast through their CPI motifs, which also allosterically modulate capping strength. In contrast, twinfilin works downstream of CP recruitment, regulating the turnover of CP through its CPI motif and a non-allosteric mechanism. Collectively, our findings reveal how three CPI motif proteins work together to regulate CP in a stepwise fashion during endocytosis.

Utilizing chemically induced dimerization of FKBP to analyze endocytosis by live-cell imaging in budding yeast.

Chemically induced dimerization (CID) is a useful tool for artificially inducing protein-protein interactions. Although CID has been used extensively for live-cell microscopy applications in mammalian systems, it is rarely utilized in yeast cell biology studies. Here, we present a step-by-step protocol for the utilization of a CID system in live-cell microscopy experiments of budding yeast endocytosis. While focusing on the study of endocytosis, this protocol framework is adaptable to the study of other cellular processes in Saccharomyces cerevisiae. For complete details on the use and execution of this protocol, please refer to Lamb et al. (2021).

The dynein light chain protein Tda2 functions as a dimerization engine to regulate actin capping protein during endocytosis.

Clathrin- and actin-mediated endocytosis is a fundamental process in eukaryotic cells. Previously, we discovered Tda2 as a new yeast dynein light chain (DLC) that works with Aim21 to regulate actin assembly during endocytosis. Here we show Tda2 functions as a dimerization engine bringing two Aim21 molecules together using a novel binding surface different than the canonical DLC ligand binding groove. Point mutations on either protein that diminish the Tda2-Aim21 interaction in vitro cause the same in vivo phenotype as TDA2 deletion showing reduced actin capping protein (CP) recruitment and increased filamentous actin at endocytic sites. Remarkably, chemically induced dimerization of Aim21 rescues the endocytic phenotype of TDA2 deletion. We also uncovered a CP interacting motif in Aim21, expanding its function to a fundamental cellular pathway and showing such motif exists outside mammalian cells. Furthermore, specific disruption of this motif causes the same deficit of actin CP recruitment and increased filamentous actin at endocytic sites as AIM21 deletion. Thus, the data indicate the Tda2-Aim21 complex functions in actin assembly primarily through CP regulation. Collectively, our results provide a mechanistic view of the Tda2-Aim21 complex and its function in actin network regulation at endocytic sites.

Composition-based prediction and rational manipulation of prion-like domain recruitment to stress granules.

Mutations in a number of stress granule-associated proteins have been linked to various neurodegenerative diseases. Several of these mutations are found in aggregation-prone prion-like domains (PrLDs) within these proteins. In this work, we examine the sequence features governing PrLD localization to stress granules upon stress. We demonstrate that many yeast PrLDs are sufficient for stress-induced assembly into microscopically visible foci that colocalize with stress granule markers. Additionally, compositional biases exist among PrLDs that assemble upon stress, and these biases are consistent across different stressors. Using these biases, we have developed a composition-based prediction method that accurately predicts PrLD assembly into foci upon heat shock. We show that compositional changes alter PrLD assembly behavior in a predictable manner, while scrambling primary sequence has little effect on PrLD assembly and recruitment to stress granules. Furthermore, we were able to design synthetic PrLDs that were efficiently recruited to stress granules, and found that aromatic amino acids, which have previously been linked to PrLD phase separation, were dispensable for this recruitment. These results highlight the flexible sequence requirements for stress granule recruitment and suggest that PrLD localization to stress granules is driven primarily by amino acid composition, rather than primary sequence.

Preimmunization with a heat-killed preparation of Mycobacterium vaccae enhances fear extinction in the fear-potentiated startle paradigm.

The hygiene hypothesis or "Old Friends" hypothesis proposes that inflammatory diseases are increasing in modern urban societies, due in part to reduced exposure to microorganisms that drive immunoregulatory circuits, and a failure to terminate inappropriate inflammatory responses. Inappropriate inflammation is also emerging as a risk factor for trauma-related, anxiety, and affective disorders, including posttraumatic stress disorder (PTSD), which is characterized as persistent re-experiencing of the trauma after a traumatic experience. Traumatic experiences can lead to long-lasting fear memories and exaggerated fear potentiation of the acoustic startle reflex. The acoustic startle reflex is an ethologically relevant reflex and can be potentiated in both humans and rats through Pavlovian conditioning. Mycobacterium vaccae NCTC 11659 is a soil-derived bacterium with immunoregulatory and anti-inflammatory properties that has been demonstrated to confer stress resilience in mice. Here we immunized adult male Sprague Dawley rats 3×, once per week, with a heat-killed preparation of M. vaccae NCTC 11659 (0.1mg, s.c., in 100µl borate-buffered saline) or vehicle, and, then, 3weeks following the final immunization, tested them in the fear-potentiated startle paradigm; controls were maintained under home cage control conditions throughout the experiment (n=11-12 per group). Rats were tested on days 1 and 2 for baseline acoustic startle, received fear conditioning on days 3 and 4, and underwent fear extinction training on days 5-10. Rats were euthanized on day 11 and brain tissue was sectioned for analysis of mRNA expression for genes important in control of brain serotonergic signaling, including tph2, htr1a, slc6a4, and slc22a3, throughout the brainstem dorsal and median raphe nuclei. Immunization with M. vaccae had no effect on baseline acoustic startle or fear expression on day 5. However, M. vaccae-immunized rats showed enhanced between-session and within-session extinction on day 6, relative to vehicle-immunized controls. Immunization with M. vaccae and fear-potentiated startle altered serotonergic gene expression in a gene- and subregion-specific manner. These data are consistent with the hypothesis that immunoregulatory strategies, such as preimmunization with M. vaccae, have potential for prevention of stress- and trauma-related psychiatric disorders.

Novel function of a dynein light chain in actin assembly during clathrin-mediated endocytosis.

Clathrin- and actin-mediated endocytosis is essential in eukaryotic cells. In this study, we demonstrate that Tda2 is a novel protein of the endocytic machinery necessary for normal internalization of native cargo in yeast. Tda2 has not been classified in any protein family. Unexpectedly, solving the crystal structure of Tda2 revealed it belongs to the dynein light chain family. However, Tda2 works independently of the dynein motor complex and microtubules. Tda2 forms a tight complex with the polyproline motif-rich protein Aim21, which interacts physically with the SH3 domain of the Arp2/3 complex regulator Bbc1. The Tda2-Aim21 complex localizes to endocytic sites in a Bbc1- and filamentous actin-dependent manner. Importantly, the Tda2-Aim21 complex interacts directly with and facilitates the recruitment of actin-capping protein, revealing barbed-end filament capping at endocytic sites to be a regulated event. Thus, we have uncovered a new layer of regulation of the actin cytoskeleton by a member of a conserved protein family that has not been previously associated with a function in endocytosis.