Biology subjects

Raczkowski, A. M.

Publications and source records attributed to Raczkowski, A. M..

5 recordsLinked to original sources

bioRxiv2024

Structural basis for catalysis and selectivity of phospholipid synthesis by eukaryotic choline-phosphotransferase

Phospholipids are the most abundant component in lipid membranes and are essential for the structural and functional integrity of the cell. In eukaryotic cells, phospholipids are primarily synthesized de novo through the Kennedy pathway that involves multiple enzymatic processes. The terminal reaction is mediated by a group of cytidine-5-diphosphate (CDP)-choline /CDP-ethanolamine-phosphotransferases (CPT/EPT) that use 1,2-diacylglycerol (DAG) and CDP-choline or CDP-ethanolamine to produce phosphatidylcholine (PC) or phosphatidylethanolamine (PE) those are the main phospholipids in eukaryotic cells. Here we present the structure of the yeast CPT1 in multiple substrate-bound states. Structural and functional analysis of these binding-sites reveal the critical residues for the DAG acyl-chain preference and the choline/ethanolamine selectivity. Additionally, we present the structure in complex with a potent inhibitor characterized in this study. The ensemble of structures allows us to propose the reaction mechanism for phospholipid biosynthesis by the family of CDP-alcohol phosphatidyltransferases (CDP-APs).

biochemistry↗

bioRxiv2021

Numb is required for optimal contraction of skeletal muscle

BackgroundThe role of Numb, a protein that is important for cell fate and development was investigated in adult skeletal muscle in mice using a conditional, inducible knockout (cKO) model. MethodsNumb expression was evaluated by Western blot. Numb localization was determined by confocal microscopy. The effects of cKO of Numb and the closely-related gene Numb-like in skeletal muscle fibers was evaluated by in-situ physiology; transmission and focused ion beam scanning electron microscopy; 3-dimensional reconstruction of mitochondrial; lipidomics; and bulk RNA-sequencing. Additional studies using primary mouse myotubes investigated the effects the effects of Numb knockdown on cell fusion, mitochondrial function and calcium transients. ResultsNumb protein expression was reduced by [~]70% (p < 0.01) at 24 as compared to 3 months of age. Numb was localized within muscle fibers as bands traversing fibers at regularly spaced intervals in close proximity to dihydropyridine receptors. The cKO of Numb and Numb-like reduced specific tetanic force by 36%, p < 0.01), altered mitochondrial spatial relationships to sarcomeric structures, increased Z-line spacing by 30% (p < 0.0001), perturbed sarcoplasmic reticulum organization and reduced mitochondrial volume by over 80% (p < 0.01). Only six genes were differentially expressed in cKO mice: Itga4, Sema7a, Irgm2, Vezf1, Mib1 and Tmem132a. Several lipid mediators derived from polyunsaturated fatty acid (PUFAs) through lipoxygenases were upregulated in Numb cKO skeletal muscle; 12-HEPE was increased by [~]250% (p < 0.05) and 17,18-EpETE by [~]240% (p < 0.05). In mouse primary myotubes, Numb knock-down reduced cell fusion ([~]20%, p < 0.01) and mitochondrial function and delayed the caffeine-induced rise in cytosolic calcium concentrations by more than 100% (p < 0.01). ConclusionsThese findings implicate Numb as a critical factor in skeletal muscle structure and function which appear to be critical for calcium release; we therefore speculate Numb plays critical roles in excitation-contraction coupling, one of the putative targets of aged skeletal muscles. These findings provide new insights into the molecular underpinnings of the loss of muscle function observed with sarcopenia.

physiology↗

bioRxiv2020

Waffle method: A general and flexible approach for FIB-milling small and anisotropically oriented samples

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens that do not vitrify well, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and cellular specimens that distribute poorly across grid squares. Here we present a general approach we call the Waffle Method which leverages high-pressure freezing to address these challenges. We illustrate the mitigation of these challenges by applying the Waffle Method and cryo-ET to reveal the macrostructure of the polar tube in microsporidian spores in multiple complementary orientations, which was previously not possible due to preferred orientation of the spores on the grid. We demonstrate the broadness of the Waffle Method by applying it to three additional cellular samples and a single particle sample using a variety of cryo-FIB-milling hardware, with both manual and automated approaches. We also present a unique and critical stress-relief gap designed specifically for waffled lamellae. Additionally, we describe applications of the Waffle Method which are currently being explored. We propose the Waffle Method as a way to achieve many of the advantages of cryo-liftout on the specimen grid while avoiding the long, challenging, and technically-demanding process required for cryo-liftout.

microbiology↗

bioRxiv2019

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over one million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides - arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9 [A] resolution structure of M. abscessus AftD determined by single particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4 and 3.5 [A] structures of a mutant with impaired ACP binding reveal a conformational change that suggests the ACP may regulate AftD function. Using a conditional knock-out constructed in M. smegmatis, mutagenesis experiments confirm the essentiality of the putative active site and the ACP binding for AftD function.

biochemistry↗

bioRxiv2018

Benchmarking cryo-EM single particle analysis workflow

Cryo electron microscopy facilities running multiple instruments and serving users with varying skill levels need a robust and reliable method for benchmarking both the hardware and software components of their single particle analysis workflow. The workflow is complex, with many bottlenecks existing at the specimen preparation, data collection and image analysis steps; the samples and grid preparation can be of unpredictable quality, there are many different protocols for microscope and camera settings, and there is a myriad of software programs for analysis that can depend on dozens of settings chosen by the user. For this reason, we believe it is important to benchmark the entire workflow, using a standard sample and standard operating procedures, on a regular basis. This provides confidence that all aspects of the pipeline are capable of producing maps to high resolution. Here we describe benchmarking procedures using a test sample, rabbit muscle aldolase.

molecular biology↗