bims-lypmec 2024-04-07 papers

Anal Chim Acta. 2024 May 08. pii: S0003-2670(24)00307-6. [Epub ahead of print]1302 342506

Exquisite visualization of mitophagy and monitoring the increase of lysosomal micro-viscosity in mitophagy with an unusual pH-independent lysosomal rotor.

Rui Yang, Wei He, Changxin Zhu, Xifeng Yang, Yawei Kuang, Tao Zhu, Jingyang Xu, Yuang Zhao, Tingwang Jiang, Yushen Liu, Mengmeng Wei.

BACKGROUND: Mitophagy plays indispensable roles in maintaining intracellular homeostasis in most eukaryotic cells by selectively eliminating superfluous components or damaged organelles. Thus, the co-operation of mitochondrial probes and lysosomal probes was presented to directly monitor mitophagy in dual colors. Nowadays, most of the lysosomal probes are composed of groups sensitive to pH, such as morpholine, amine and other weak bases. However, the pH in lysosomes would fluctuate in the process of mitophagy, leading to the optical interference. Thus, it is crucial to develop a pH-insensitive probe to overcome this tough problem to achieve exquisite visualization of mitophagy.
RESULTS: In this study, we rationally prepared a pH-independent lysosome probe to reduce the optical interference in mitophagy, and thus the process of mitophagy could be directly monitored in dual color through cooperation between IVDI and MTR, depending on Förster resonance energy transfer mechanism. IVDI shows remarkable fluorescence enhancement toward the increase of viscosity, and the fluorescence barely changes when pH varies. Due to the sensitivity to viscosity, the probe can visualize micro-viscosity alterations in lysosomes without washing procedures, and it showed better imaging properties than LTR. Thanks to the inertia of IVDI to pH, IVDI can exquisitely monitor mitophagy with MTR by FRET mechanism despite the changes of lysosomal pH in mitophagy, and the reduced fluorescence intensity ratio of green and red channels can indicate the occurrence of mitophagy. Based on the properties mentioned above, the real-time increase of micro-viscosity in lysosomes during mitophagy was exquisitely monitored through employing IVDI.
SIGNIFICANCE AND NOVELTY: Compared with the lysosomal fluorescent probes sensitive to pH, the pH-inert probe could reduce the influence of pH variation during mitophagy to achieve exquisite visualization of mitophagy in real-time. Besides, the probe could monitor the increase of lysosomal micro-viscosity in mitophagy. So, the probe possesses tremendous potential in the visualization of dynamic changes related to lysosomes in various physiological processes.

Keywords: Exquisitely long-term visualization; FRET mechanism; Lysosomal rotor; Mitophagy; pH-independent

DOI: https://doi.org/10.1016/j.aca.2024.342506

J Cell Sci. 2024 Apr 05. pii: jcs.261765. [Epub ahead of print]

Collapse of late endosomal pH elicits a rapid Rab7 response via V-ATPase and RILP.

R J Mulligan, M M Magaj, L Digilio, S Redemann, C C Yap, B Winckler.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of proper pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). We used the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyper-activation of Rab7 and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR) recycling on pH-neutralized LEs. Either pH neutralization (NH4Cl) or Rab7 hyper-active mutants alone can phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds or in disease states.

Keywords: Acidification; Late endosome; Lysosomal membrane permeabilization; Membrane trafficking; RILP; Rab7; V-ATPase

DOI: https://doi.org/10.1242/jcs.261765

ACS Omega. 2024 Mar 26. 9(12): 13494-13508

Review on Lysosomal Metal Ion Detection Using Fluorescent Probes.

Akshay Silswal, Kavyashree P, Apurba Lal Koner.

Metal ions are indispensable and play an important role in living systems. Metal ions coordinated to metalloenzymes pocket activate the bound substrate and labile metal ions maintaining the ionic balance. The amount of metal ions present in various subcellular compartments of the cells is highly regulated for maintaining cellular homeostasis. An imbalance in the metal ion concentration is related to several diseases and results in serious pathological conditions. Mostly the internalized metal ions are processed in the lysosomal compartment of the cell. A delicate regulation of metal ions in the lysosomal compartment can modulate the lysosomal pH and inhibit hydrolytic enzymes, which ultimately causes lysosomal storage disorders. In the past decade, the understanding and regulation of lysosomal metal ions based on fluorometric methods have gained significant attention. In this review, we have comprehensively summarized the development of various fluorescent reporters over the past five years for a selective and sensitive estimation of lysosomal metal ion concentration. We believe this consolidated and timely review will help researchers working in the areas associated with lysosomal metal ions.

DOI: https://doi.org/10.1021/acsomega.3c08606

Front Physiol. 2024 ;15 1294369

The cardiovascular changes underlying a low cardiac output with exercise in patients with type 2 diabetes mellitus.

Per Lav Madsen, Casper Sejersen, Michael Nyberg, Martin Heyn Sørensen, Ylva Hellsten, Peter Gaede, Annemie Stege Bojer.

The significant morbidity and premature mortality of type 2 diabetes mellitus (T2DM) is largely associated with its cardiovascular consequences. Focus has long been on the arterial atheromatosis of DM giving rise to early stroke and myocardial infarctions, whereas less attention has been given to its non-ischemic cardiovascular consequences. Irrespective of ischemic changes, T2DM is associated with heart failure (HF) most commonly with preserved ejection fraction (HFpEF). Largely due to increasing population ages, hypertension, obesity and T2DM, HFpEF is becoming the most prevalent form of heart failure. Unfortunately, randomized controlled trials of HFpEF have largely been futile, and it now seems logical to address the important different phenotypes of HFpEF to understand their underlying pathophysiology. In the early phases, HFpEF is associated with a significantly impaired ability to increase cardiac output with exercise. The lowered cardiac output with exercise results from both cardiac and peripheral causes. T2DM is associated with left ventricular (LV) diastolic dysfunction based on LV hypertrophy with myocardial disperse fibrosis and significantly impaired ability for myocardial blood flow increments with exercise. T2DM is also associated with impaired ability for skeletal muscle vasodilation during exercise, and as is the case in the myocardium, such changes may be related to vascular rarefaction. The present review discusses the underlying phenotypical changes of the heart and peripheral vascular system and their importance for an adequate increase in cardiac output. Since many of the described cardiovascular changes with T2DM must be considered difficult to change if fully developed, it is suggested that patients with T2DM are early evaluated with respect to their cardiovascular compromise.

Keywords: cardiac fibrosis; exercise intolerance; heart failure with a preserved ejection fraction; hypertension; myocardial blood flow and flow reserve; type 2 diabetes mellitus

DOI: https://doi.org/10.3389/fphys.2024.1294369

Nat Commun. 2024 Mar 30. 15(1): 2793

Membrane fission via transmembrane contact.

Russell K W Spencer, Isaac Santos-Pérez, Izaro Rodríguez-Renovales, Juan Manuel Martinez Galvez, Anna V Shnyrova, Marcus Müller.

Division of intracellular organelles often correlates with additional membrane wrapping, e.g., by the endoplasmic reticulum or the outer mitochondrial membrane. Such wrapping plays a vital role in proteome and lipidome organization. However, how an extra membrane impacts the mechanics of the division has not been investigated. Here we combine fluorescence and cryo-electron microscopy experiments with self-consistent field theory to explore the stress-induced instabilities imposed by membrane wrapping in a simple double-membrane tubular system. We find that, at physiologically relevant conditions, the outer membrane facilitates an alternative pathway for the inner-tube fission through the formation of a transient contact (hemi-fusion) between both membranes. A detailed molecular theory of the fission pathways in the double membrane system reveals the topological complexity of the process, resulting both in leaky and leakless intermediates, with energies and topologies predicting physiological events.

DOI: https://doi.org/10.1038/s41467-024-47122-w