Biology subjects

Feldman, J. L.

Publications and source records attributed to Feldman, J. L..

5 recordsLinked to original sources

bioRxiv2018

A two-step mechanism for the inactivation of microtubule organizing center function at the centrosome

SummaryDuring mitosis, the centrosome acts as a microtubule organizing center (MTOC), orchestrating microtubules into the mitotic spindle through its pericentriolar material (PCM). This activity is biphasic, cycling through assembly and disassembly during the cell cycle. Although hyperactive centrosomal MTOC activity is a hallmark of some cancers, little is known about how the centrosome is inactivated as an MTOC. Analysis of endogenous PCM proteins in C. elegans revealed that the PCM is composed of distinct protein territories that are removed from the centrosome at different rates and using different behaviors. Inhibition of PP2A phosphatases stabilized the PCM and perturbation of cortical pulling forces altered the timing and behavior by which proteins were removed from the centrosome. These data indicate that PCM disassembly is a two-step process, beginning with a phosphatase-dependent dissolution of PCM proteins followed by the ejection of ruptured PCM by cortical forces, ultimately inactivating MTOC function at the centrosome.

cell biology↗

bioRxiv2018

Tissue-specific degradation of essential centrosome components reveals distinct microtubule populations at microtubule organizing centers

Non-centrosomal microtubule organizing centers (ncMTOCs) are found in most differentiated cells, but how these structures regulate microtubule organization and dynamics is largely unknown. We optimized a tissue-specific degradation system to test the role of the essential centrosomal microtubule nucleators {gamma}-tubulin ring complex ({gamma}-TURC) and AIR-1/Aurora A at the apical ncMTOC, where they both localize in C. elegans embryonic intestinal epithelial cells. As at the centrosome, the core {gamma}-TURC component GIP-1/GCP3 is required to recruit other {gamma}-TuRC components to the apical ncMTOC including MZT-1/MZT1, characterized here for the first time in animal development. In contrast, AIR-1 and MZT-1 were specifically required to recruit {gamma}-TuRC to the centrosome, but not to centrioles or to the apical ncMTOC. Surprisingly, microtubules remain robustly organized at the apical ncMTOC upon {gamma}-TuRC and AIR-1 co-depletion, and upon depletion of other known microtubule regulators including TPXL-1/TPX2, ZYG-9/chTOG, PTRN-1/CAMSAP, and NOCA-1/Ninein. However, loss of GIP-1 removed a subset of dynamic EBP-2/EB1-marked microtubules, and the remaining dynamic microtubules grew faster. Together, these results suggest that different MTOCs use discrete proteins for their function, and that the apical ncMTOC is composed of distinct populations of {gamma}-TuRC-dependent and independent microtubules that compete for a limited pool of resources.

cell biology↗

bioRxiv2017

Directed evolution of TurboID for efficient proximity labeling in living cells and organisms

Protein interaction networks and protein compartmentation underlie every signaling process and regulatory mechanism in cells. Recently, proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, the two enzymes commonly used for PL come with tradeoffs - BioID is slow, requiring tagging times of 18-24 hours, while APEX peroxidase uses substrates that have limited cell permeability and high toxicity. To address these problems, we used yeast display-based directed evolution to engineer two mutants of biotin ligase, TurboID and miniTurbo, with much greater catalytic efficiency than BioID, and the ability to carry out PL in cells in much shorter time windows (as little as 10 minutes) with non-toxic and easily deliverable biotin. In addition to shortening PL time by 100-fold and increasing PL yield in cell culture, TurboID enabled biotin-based PL in new settings, including yeast, Drosophila, and C. elegans.

bioengineering↗

bioRxiv2017

Inhaled nicotine equivalent to cigarette smoking disrupts systemic and uterine hemodynamics and induces cardiac arrhythmia in pregnant rats

Maternal smoking with obligatory nicotine inhalation is associated with preterm delivery, low birth weight, fetal growth retardation and developmental defects. We tested the hypothesis that cigarette smoking-relevant nicotine inhalation during pregnancy impairs cardiovascular function and uterine hemodynamics with consequential fetal ischemia. Pregnant rats exposed to episodic inhaled nicotine via a novel lung alveolar region-targeted aerosol method produced nicotine pharmacokinetics resembling cigarette smoking in humans. This clinically relevant nicotine aerosol inhalation (NAI) induced transient reduction and irregular fluctuations in uterine artery blood flow associated with cardiac arrhythmia and high magnitude irregular fluctuations of systemic blood pressure. The arrhythmia included sinoatrial (SA) block, sinus arrest, 2o and 3o atrioventricular (A-V) block and supraventricular escape rhythm. These effects were blocked by the nicotinic receptor (nAChR) antagonist mecamylamine. Resection of the ovarian nerve, which innervates uterine blood vessels, counteracted the NAI-induced reduction in uterine blood flow. We suggest that the rapid rise pattern of arterial blood nicotine concentration stimulates and then desensitizes autonomic nAChRs leading to disruptions of cardiac function as well as systemic and uterine hemodynamics that reduces uteroplacental blood flow, a mechanism underlying maternal smoking-associated pregnancy complications and developmental disorders. These findings challenge the safety of pure nicotine inhalation, i.e., E-cigarettes.

pharmacology and toxicology↗

bioRxiv2017

Chronic intermittent nicotine delivery with lung alveolar region-targeted aerosol technology produces circadian blood pharmacokinetics in rats resembling human smokers

IntroductionCigarette smoke is an aerosol containing microparticles that carry nicotine into lung alveolar region where nicotine is rapidly absorbed into circulation. Nicotine exposure in smokers is a chronic intermittent process, with intake during wakefulness and abstinence during sleep resulting in circadian fluctuation of blood nicotine levels. Here we present a smoking-relevant nicotine exposure device and rodent model.\n\nMethodsWe developed a computer controlled integrated platform where freely moving rodents can be exposed to episodic nicotine aerosol on an investigator-designed schedule. Rats were exposed to nicotine aerosol once every half hr in the dark phase of 12/12-hr dark/light cycles for 10 days. Plasma nicotine and its metabolite cotinine levels were determined with a LC-MS/MS method.\n\nResultsWe characterized the aerosol in the breathing zone of the rodent exposure chamber. The droplet size distribution was within the respirable diameter range. The system can generate a wide range of nicotine concentrations in air that meet a variety of experimental needs. We optimized the parameters of aerosol generation and exposure: plasma nicotine and cotinine concentrations reached 30-35 ng/ml and 190-240 ng/ml, respectively. The nicotine levels and circadian patterns resembled the pharmacokinetic pattern of human smokers.\n\nConclusionsWe developed an aerosol system that can produce chronic intermittent nicotine exposure in unanesthetized and unrestrained rodents with route of administration and circadian blood pharmacokinetics resembling human smokers. This methodology is a novel tool for studies of behavior, pharmacology and toxicology of chronic nicotine exposure, nicotine addiction, tobacco-related diseases, teratogenicity, and for discovery of therapeutics.\n\nImplicationsWe developed a method and an alveolar region-targeted aerosol system that provides chronic intermittent nicotine exposure in rodents. The method produces clinically relevant animal models with the route of administration and circadian pharmacokinetics resembling human smokers. This method is a novel tool for understanding the health effects of chronic nicotine exposures such as with tobacco cigarettes, E-cigarettes and other tobacco products, for studies of pharmacology, toxicology, nicotine addiction, tobacco-related diseases, and for discovery of medications.

pharmacology and toxicology↗