Novel regulatory roles of endocytic membrane trafficking proteins in mitochondrial homeostasis

Endocytic membrane trafficking is a basic cell process that is critical for regulating the transport of lipids and proteins. Our lab focuses on the cellular functions and mechanisms of the proteins that regulate these pathways. A key family of regulatory proteins is the C-terminal Eps15 Homology Domain (EHD) protein family. The EHD family includes EHD1-4, which are ubiquitously expressed in mammalian tissues. While these isoforms do have some overlapping functions, each protein also has distinct activities in regulating the shape and fission of membranes throughout the endocytic pathways. Specifically, EHD1 uses ATP hydrolysis to induce constriction and fission of endocytic membranes. EHD1 is recruited to tubular recycling endosomes (TREs) by interacting with Molecules Interacting with CAsL-Like 1 (MICAL-L1) and it performs fission to release cargo-containing vesicles from the TRE. Our lab demonstrated that upon EHD1 depletion, the TREs become elongated due to the lack of fission and the receptors that recycle through this pathway display impaired recycling to the plasma membrane. Furthermore, our lab and others have shown that EHD1 not only interacts with MICAL-L1, but also with a variety of other proteins, such as the retromer cargo selection complex (CSC), which is known to regulate the trafficking of membranes iii and proteins between endosomes and the Golgi complex. Recently, the proposed role of VPS35, a core protein of the retromer complex, has expanded, and it was found to interact with and control the mitochondrial fission protein, Drp1. However, the connection between EHD1 and the retromer and their role in mitochondrial homeostasis is less clear. It was previously thought that endocytic regulatory proteins exclusively impacted membrane trafficking pathways, but recent studies suggest that endocytic regulatory proteins play a role in many other pathways including ciliogenesis, centrosome disengagement, and mitochondrial homeostasis. Herein, I describe a novel role for in endocytic regulatory proteins in controlling mitochondrial fission and mitochondrial-induced apoptosis. My studies led to a model by which EHD1 regulates the localization of the retromer within the cell; accordingly, when EHD1 is absent, the retromer no longer regulates the mitochondrial fission protein, Drp1. In addition, I demonstrate for the first time, a connection between endocytic proteins and apoptosis by proposing a model for an expanded role for the retromer complex in regulating mitochondrial-induced apoptosis through the trafficking of the anti-apoptotic protein, Bcl-xL

Real-Time Measurement of Herbicides in the Atmosphere: A Case Study of MCPA and 2,4-D during Field Application

Atmospheric sources of herbicides enable short- and long-range transport of these compounds to off-target areas but the concentrations and mechanisms are poorly understood due, in part, to the challenge of detecting these compounds in the atmosphere. We present chemical ionization time-of-flight mass spectrometry as a sensitive, real-time technique to detect chlorinated phenoxy acid herbicides in the atmosphere, using measurements during and after application over a field at Colorado State University as a case study. Gas-phase 2,4-dichlorophenoxyacetic acid (2,4-D) mixing ratios were greatest during application (up to 20 pptv), consistent with rapid volatilization from spray droplets. In contrast, atmospheric concentrations of 2-methyl-4-chlorophenoxyacetic acid (MCPA) increased for several hours after the initial application, indicative of a slower source than 2,4-D. The maximum observed gas-phase MCPA was 60 pptv, consistent with a post-application volatilization source to the atmosphere. Exposure to applied pesticides in the gas-phase can thus occur both during and at least several hours after application. Spray droplet volatilization and direct volatilization from surfaces may both contribute pesticides to the atmosphere, enabling pesticide transport to off-target and remote regions

Atmospheric OH Oxidation of Three Chlorinated Aromatic Herbicides

Chlorinated phenoxy acids are a widely used class of herbicides and have been found in remote regions far from sources. However, the atmospheric chemistry of these compounds is poorly understood. We use an oxidative flow reactor coupled to chemical ionization mass spectrometry to investigate OH oxidation of two chlorinated phenoxyacid herbicides (2-methyl-4-chlorophenoxyacetic acid (MCPA) and mecoprop-p) and one chlorinated pyridine herbicide (triclopyr). OH radicals add to the aromatic rings of the three herbicides, produce peroxides via hydrogen abstraction, or fragment at the ether bond. OH oxidation of MCPA produced two potentially toxic compounds: chlorosalicylaldehyde and chlorosalicylic acid. We use standards to validate the detection of these oxidation products by acetate CIMS and quantify the reaction rate. Oxidation of triclopyr produced a known endocrine disruptor, 3,5,6-trichloro-2-pyridinol. Thus, while some OH oxidation products are less toxic than the parent molecules (e.g., C₁-₅ carboxylic acids), others may be as or more toxic than the parent herbicide. We determine effective rate coefficients for OH addition to the aromatic ring (<i>k</i>_OH) for mecoprop-p of 1.5 (±0.7) × 10^–12 cm³ molecules^–1 s^–1 and for MCPA of 2.6 (±0.3) × 10^–12 cm³ molecules^–1 s^–1. The atmospheric lifetimes with respect to OH are thus long enough that photochemistry may be relevant to the environmental fate of these pesticides

Vesicular trafficking plays a role in centriole disengagement and duplication

Centrosomes are the major microtubule-nucleating and microtubule-organizing centers of cells and play crucial roles in microtubule anchoring, organelle positioning, and ciliogenesis. At the centrosome core lies a tightly associated or "engaged" mother-daughter centriole pair. During mitotic exit, removal of centrosomal proteins pericentrin and Cep215 promotes "disengagement" by the dissolution of intercentriolar linkers, ensuring a single centriole duplication event per cell cycle. Herein, we explore a new mechanism involving vesicular trafficking for the removal of centrosomal Cep215. Using small interfering RNA and CRISPR/ Cas9 gene-edited cells, we show that the endocytic protein EHD1 regulates Cep215 transport from centrosomes to the spindle midbody, thus facilitating disengagement and duplication. We demonstrate that EHD1 and Cep215 interact and show that Cep215 displays increased localization to vesicles containing EHD1 during mitosis. Moreover, Cep215-containing vesicles are positive for internalized transferrin, demonstrating their endocytic origin. Thus, we describe a novel relationship between endocytic trafficking and the centrosome cycle, whereby vesicles of endocytic origin are used to remove key regulatory proteins from centrosomes to control centriole duplication.National Institute of General Medical Sciences (NIGMS) [R01GM074876, P30GM106397]; National Cancer Institute [P30 CA23074]; NIH/NIGMS [R01GFM110166, R01GM126035]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]