Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in intact isolated yeast mitochondria using photoactivated localization microscopy with sub-diffraction limited spatial precision, we demonstrate that Tom40 movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole

Activation of H+-ATPase of the Plasma Membrane of Saccharomyces cerevisiae by Glucose: The Role of Sphingolipid and Lateral Enzyme Mobility

Activation of the plasma membrane H+-ATPase of the yeast Saccharomyces cerevisiae by glucose is a complex process that has not yet been completely elucidated. This study aimed to shed light on the role of lipids and the lateral mobility of the enzyme complex during its activation by glucose. The significance of H+-ATPase oligomerization for the activation of H+-ATPase by glucose was shown using the strains lcb1-100 and erg6, with the disturbed synthesis of sphyngolipid and ergosterol, respectively. Experiments with GFP-fused H+-ATPase showed a decrease in fluorescence anisotropy during the course of glucose activation, suggesting structural reorganization of the molecular domains. An immunogold assay showed that the incubation with glucose results in the spatial redistribution of ATPase complexes in the plasma membrane. The data suggest that (1) to be activated by glucose, H+-ATPase is supposed to be in an oligomeric state, and (2) glucose activation is accompanied by the spatial movements of H+-ATPase clusters in the PM

Hostile Takeover by Plasmodium: Reorganization of Parasite and Host Cell Membranes during Liver Stage Egress

The protozoan parasite Plasmodium is transmitted by female Anopheles mosquitoes and undergoes obligatory development within a parasitophorous vacuole in hepatocytes before it is released into the bloodstream. The transition to the blood stage was previously shown to involve the packaging of exoerythrocytic merozoites into membrane-surrounded vesicles, called merosomes, which are delivered directly into liver sinusoids. However, it was unclear whether the membrane of these merosomes was derived from the parasite membrane, the parasitophorous vacuole membrane or the host cell membrane. This knowledge is required to determine how phagocytes will be directed against merosomes. Here, we fluorescently label the candidate membranes and use live cell imaging to show that the merosome membrane derives from the host cell membrane. We also demonstrate that proteins in the host cell membrane are lost during merozoite liberation from the parasitophorous vacuole. Immediately after the breakdown of the parasitophorous vacuole membrane, the host cell mitochondria begin to degenerate and protein biosynthesis arrests. The intact host cell plasma membrane surrounding merosomes allows Plasmodium to mask itself from the host immune system and bypass the numerous Kupffer cells on its way into the bloodstream. This represents an effective strategy for evading host defenses before establishing a blood stage infection

SLC35A2â CDG: Functional characterization, expanded molecular, clinical, and biochemical phenotypes of 30 unreported Individuals

Pathogenic de novo variants in the Xâ linked gene SLC35A2 encoding the major Golgiâ localized UDPâ galactose transporter required for proper protein and lipid glycosylation cause a rare type of congenital disorder of glycosylation known as SLC35A2â congenital disorders of glycosylation (CDG; formerly CDGâ IIm). To date, 29 unique de novo variants from 32 unrelated individuals have been described in the literature. The majority of affected individuals are primarily characterized by varying degrees of neurological impairments with or without skeletal abnormalities. Surprisingly, most affected individuals do not show abnormalities in serum transferrin Nâ glycosylation, a common biomarker for most types of CDG. Here we present data characterizing 30 individuals and add 26 new variants, the single largest study involving SLC35A2â CDG. The great majority of these individuals had normal transferrin glycosylation. In addition, expanding the molecular and clinical spectrum of this rare disorder, we developed a robust and reliable biochemical assay to assess SLC35A2â dependent UDPâ galactose transport activity in primary fibroblasts. Finally, we show that transport activity is directly correlated to the ratio of wildâ type to mutant alleles in fibroblasts from affected individuals.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/1/humu23731_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/2/humu23731-sup-0001-Supp_Mat__2019.2.10_.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150498/3/humu23731.pd

Attomolar Detection of Botulinum Toxin Type A in Complex Biological Matrices

BACKGROUND: A highly sensitive, rapid and cost efficient method that can detect active botulinum neurotoxin (BoNT) in complex biological samples such as foods or serum is desired in order to 1) counter the potential bioterrorist threat 2) enhance food safety 3) enable future pharmacokinetic studies in medical applications that utilize BoNTs. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a botulinum neurotoxin serotype A assay with a large immuno-sorbent surface area (BoNT/A ALISSA) that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate. In direct comparison with the "gold standard" mouse bioassay, the ALISSA is four to five orders of magnitudes more sensitive and considerably faster. Our method reaches attomolar sensitivities in serum, milk, carrot juice, and in the diluent fluid used in the mouse assay. ALISSA has high specificity for the targeted type A toxin when tested against alternative proteases including other BoNT serotypes and trypsin, and it detects the holotoxin as well as the multi-protein complex form of BoNT/A. The assay was optimized for temperature, substrate concentration, size and volume proportions of the immuno-sorbent matrix, enrichment and reaction times. Finally, a kinetic model is presented that is consistent with the observed improvement in sensitivity. CONCLUSIONS/SIGNIFICANCE: The sensitivity, specificity, speed and simplicity of the BoNT ALISSA should make this method attractive for diagnostic, biodefense and pharmacological applications

Rapid diffusion of green fluorescent protein in the mitochondrial matrix.

It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100x objective (0.8-micron spot diam) gave half-times for fluorescence recovery of 15-19 ms with >90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2-3 x 10(-7) cm2/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid beta-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 +/- 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, approximately 0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse

Optimization of Nutrition after Brain Injury: Mechanistic and Therapeutic Considerations

Emerging science continues to establish the detrimental effects of malnutrition in acute neurological diseases such as traumatic brain injury, stroke, status epilepticus and anoxic brain injury. The primary pathological pathways responsible for secondary brain injury include neuroinflammation, catabolism, immune suppression and metabolic failure, and these are exacerbated by malnutrition. Given this, there is growing interest in novel nutritional interventions to promote neurological recovery after acute brain injury. In this review, we will describe how malnutrition impacts the biomolecular mechanisms of secondary brain injury in acute neurological disorders, and how nutritional status can be optimized in both pediatric and adult populations. We will further highlight emerging therapeutic approaches, including specialized diets that aim to resolve neuroinflammation, immunodeficiency and metabolic crisis, by providing pre-clinical and clinical evidence that their use promotes neurologic recovery. Using nutrition as a targeted treatment is appealing for several reasons that will be discussed. Given the high mortality and both short- and long-term morbidity associated with acute brain injuries, novel translational and clinical approaches are needed