Kristyn Gumpper

MG53-mediated cell membrane repair protects against acute kidney injury

Recombinant MG53 translocates to sites of injury in the proximal tubule of the kidney and protects mice from acute kidney injury induced by ischemia or drugs. A molecular bandage for kidney injury MG53 is a protein that is primarily expressed in muscles and helps protect muscle cells from damage. Now, Duann et al. have shown that MG53 performs a similar function in the kidney as well. The authors evaluated the role of MG53 in mouse models of kidney injury induced by ischemia and reperfusion, as well as by cisplatin, a highly nephrotoxic chemotherapy drug. In each case, recombinant MG53 could be given intravenously, and the authors found that it bound to the sites of injury on kidney cells and protected them from further damage and death. MG53 treatment did not interfere with the effectiveness of cisplatin against cancer cells, suggesting that MG53 may be useful for protecting patients’ kidneys during chemotherapy. Injury to the renal proximal tubular epithelium (PTE) represents the underlying consequence of acute kidney injury (AKI) after exposure to various stressors, including nephrotoxins and ischemia/reperfusion (I/R). Although the kidney has the ability to repair itself after mild injury, insufficient repair of PTE cells may trigger inflammatory and fibrotic responses, leading to chronic renal failure. We report that MG53, a member of the TRIM family of proteins, participates in repair of injured PTE cells and protects against the development of AKI. We show that MG53 translocates to acute injury sites on PTE cells and forms a repair patch. Ablation of MG53 leads to defective membrane repair. MG53-deficient mice develop pronounced tubulointerstitial injury and increased susceptibility to I/R-induced AKI compared to wild-type mice. Recombinant human MG53 (rhMG53) protein can target injury sites on PTE cells to facilitate repair after I/R injury or nephrotoxin exposure. Moreover, in animal studies, intravenous delivery of rhMG53 ameliorates cisplatin-induced AKI without affecting the tumor suppressor efficacy of cisplatin. These findings identify MG53 as a vital component of reno-protection, and targeting MG53-mediated repair of PTE cells represents a potential approach to prevention and treatment of AKI.

3D multifocus astigmatism and compressed sensing (3D MACS) based superresolution reconstruction

Single molecule based superresolution techniques (STORM/PALM) achieve nanometer spatial resolution by integrating the temporal information of the switching dynamics of fluorophores (emitters). When emitter density is low for each frame, they are located to the nanometer resolution. However, when the emitter density rises, causing significant overlapping, it becomes increasingly difficult to accurately locate individual emitters. This is particularly apparent in three dimensional (3D) localization because of the large effective volume of the 3D point spread function (PSF). The inability to precisely locate the emitters at a high density causes poor temporal resolution of localization-based superresolution technique and significantly limits its application in 3D live cell imaging. To address this problem, we developed a 3D high-density superresolution imaging platform that allows us to precisely locate the positions of emitters, even when they are significantly overlapped in three dimensional space. Our platform involves a multi-focus system in combination with astigmatic optics and an ℓ 1-Homotopy optimization procedure. To reduce the intrinsic bias introduced by the discrete formulation of compressed sensing, we introduced a debiasing step followed by a 3D weighted centroid procedure, which not only increases the localization accuracy, but also increases the computation speed of image reconstruction. We implemented our algorithms on a graphic processing unit (GPU), which speeds up processing 10 times compared with central processing unit (CPU) implementation. We tested our method with both simulated data and experimental data of fluorescently labeled microtubules and were able to reconstruct a 3D microtubule image with 1000 frames (512×512) acquired within 20 seconds.

Fast two-dimensional super-resolution image reconstruction algorithm for ultra-high emitter density.

Single-molecule localization microscopy achieves sub-diffraction-limit resolution by localizing a sparse subset of stochastically activated emitters in each frame. Its temporal resolution is limited by the maximal emitter density that can be handled by the image reconstruction algorithms. Multiple algorithms have been developed to accurately locate the emitters even when they have significant overlaps. Currently, compressive-sensing-based algorithm (CSSTORM) achieves the highest emitter density. However, CSSTORM is extremely computationally expensive, which limits its practical application. Here, we develop a new algorithm (MempSTORM) based on two-dimensional spectrum analysis. With the same localization accuracy and recall rate, MempSTORM is 100 times faster than CSSTORM with ℓ(1)-homotopy. In addition, MempSTORM can be implemented on a GPU for parallelism, which can further increase its computational speed and make it possible for online super-resolution reconstruction of high-density emitters.

Superresolution microscope image reconstruction by spatiotemporal object decomposition and association: application in resolving t-tubule structure in skeletal muscle.

One key factor that limits resolution of single-molecule superresolution microscopy relates to the localization accuracy of the activated emitters, which is usually deteriorated by two factors. One originates from the background noise due to out-of-focus signals, sample auto-fluorescence, and camera acquisition noise; and the other is due to the low photon count of emitters at a single frame. With fast acquisition rate, the activated emitters can last multiple frames before they transiently switch off or permanently bleach. Effectively incorporating the temporal information of these emitters is critical to improve the spatial resolution. However, majority of the existing reconstruction algorithms locate the emitters frame by frame, discarding or underusing the temporal information. Here we present a new image reconstruction algorithm based on tracklets, short trajectories of the same objects. We improve the localization accuracy by associating the same emitters from multiple frames to form tracklets and by aggregating signals to enhance the signal to noise ratio. We also introduce a weighted mean-shift algorithm (WMS) to automatically detect the number of modes (emitters) in overlapping regions of tracklets so that not only well-separated single emitters but also individual emitters within multi-emitter groups can be identified and tracked. In combination with a maximum likelihood estimator method (MLE), we are able to resolve low to medium density of overlapping emitters with improved localization accuracy. We evaluate the performance of our method with both synthetic and experimental data, and show that the tracklet-based reconstruction is superior in localization accuracy, particularly for weak signals embedded in a strong background. Using this method, for the first time, we resolve the transverse tubule structure of the mammalian skeletal muscle.

Skeletal Muscle Lysosomal Function via Cathepsin Activity Measurement.

Muscle wasting or cachexia is commonly associated with aging and many diseases such as cancer, infection, autoimmune disorders, and trauma. Decrease in muscle mass, or muscle atrophy, is often caused by dysfunction of protein proteolytic systems, such as lysosomes, which regulate protein turnover and homeostasis. Lysosomes contain many hydrolases and proteases and, thus, represent the major organelle that control protein turnover. Recently, lysosomes have emerged as a signaling hub to integrate cellular functions of nutrient sensing and metabolism, autophagy, phagocytosis, and endocytosis, which are all related to tissue homeostasis. In this chapter, we describe the protocol used to measure lysosomal proteinase (cathepsins) activity in the skeletal muscle. A better understanding of lysosomal function in muscle homeostasis is critical in developing new therapeutic approaches to prevent muscle wasting.