Lysosomal Dynamics Research Articles

An increase of lysosomes through EGF-triggered endocytosis attenuated zinc-mediated lysosomal membrane permeabilization and neuronal cell death

In the context of acute brain injuries, where zinc neurotoxicity and oxidative stress are acknowledged contributors to neuronal damage, we investigated the pivotal role of lysosomes as a potential protective mechanism. Our research commenced with an exploration of epidermal growth factor (EGF) and its impact on lysosomal dynamics, particularly its neuroprotective potential against zinc-induced cytotoxicity. Using primary mouse cerebrocortical cultures, we observed the rapid induction of EGFR endocytosis triggered by EGF, resulting in a transient increase in lysosomal vesicles. Furthermore, EGF stimulated lysosomal biogenesis, evident through elevated expression of lysosomal-associated membrane protein 1 (LAMP-1) and the induction and activation of prominent lysosomal proteases, particularly cathepsin B (CTSB). This process of EGFR endocytosis was found to promote lysosomal augmentation, thus conferring protection against zinc-induced lysosomal membrane permeabilization (LMP) and subsequent neuronal death. Notably, the neuroprotective effects and lysosomal enhancement induced by EGF were almost completely reversed by the inhibition of clathrin-mediated and caveolin-mediated endocytosis pathways, along with the disruption of retrograde trafficking. Furthermore, tyrosine kinase inhibition of EGFR nullified EGFR endocytosis, resulting in the abrogation of EGF-induced lysosomal upregulation and neuroprotection. An intriguing aspect of our study is the successful replication of EGF’s neuroprotective effects through the overexpression of LAMP-1, which significantly reduced zinc-induced LMP and cell death, demonstrated in both primary mouse cerebrocortical neuronal cultures and human embryonic kidney (HEK) cells. Our research extended beyond zinc-induced neurotoxicity, as we observed EGF’s protective effects against other oxidative stressors linked to intracellular zinc release, including hydrogen peroxide (H2O2) and 1-methyl-4-phenylpyridinium ion (MPP+). Collectively, our findings unveil the intricate interplay between EGF-triggered EGFR endocytosis, lysosomal upregulation, an increase in the regulatory capacity for zinc homeostasis, and the subsequent alleviation of zinc-induced neurotoxicity. These results present promising avenues for therapeutic interventions to enhance neuroprotection by targeting lysosomal augmentation.

Diphenylbutadiene Fluorescent Analogues in Sub-Cellular Imaging and Monitoring Mitophagy.

A series of donor-acceptor (D-π-A) substituted diphenylbutadienes exhibiting solvatochromic emission and a large Stokes shift (100-200 nm) were designed and synthesized for distinctive organelle labelling, enabling real-time monitoring of organelle behaviour such as lysosomal dynamics, mitophagy monitoring, and stress responses. The morpholine-substituted D-A-D diphenylbutadiene (M2) was employed to investigate selective imaging of lysosomes, the uptake of damaged mitochondria through mitophagy, and monitoring lysosomal viscosity or pH changes. Other diphenylbutadiene derivatives (M1, M3, M4) selectively accumulated in lipid droplets. All the synthesized derivatives demonstrated significant uptake in 5-day post-fertilization zebrafish larvae, with M2 showing maximum uptake in the enterocyte-containing heart and intestinal regions, which include the lysosomes.

Dual color carbon dots for simultaneous dynamic fluorescence tracking of mitochondria and lysosomes

The development of fluorescent probes to monitor mitochondria and lysosomes will be helpful not only to realize the status monitoring of the organelles, but also to gain insights into their functions in both healthy and diseased states. Carbon Dots (CDs), as a nanomaterial with excellent properties, have provided new tools and methods for research in organelle imaging in recent years. In this study, we synthesized green fluorescence-emitting carbon dots (G-CDs) with mitochondria-targeting ability and red fluorescence-emitting carbon dots (R-CDs) with lysosome-targeting ability by using the same precursors. G-CDs are brightly with a quantum yield of up to 51.8 % and R-CDs have a long-wavelength emission of 611 nm. Based on the unique properties of G-CDs and R-CDs, we observed the changes of mitochondria during apoptosis and mitochondrial autophagy in HeLa cells by using G-CDs. And the dynamics of lysosomes in the inhibition of cellular autophagy and cellular necrosis were observed by utilizing the R-CDs. Furthermore, we observed human intestinal organoids samples stained by G-CDs and R-CDs, extending the relevant applications of carbon dots imaging to the organoids field, which brings forth new possibilities for research and drug development of related diseases.

Abstract IA-06: Elucidation of metabolic resistance mechanisms to RAS inhibition

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. We previously determined that concurrent inhibition of autophagy, using the lysosomal inhibitor chloroquine (CQ), and of ERK, using a small molecule ERK inhibitor (ERKi), synergistically suppressed the growth of pancreatic ductal adenocarcinoma (PDAC) cell lines and patient xenograft-derived (PDX) organoids in vitro and PDX tumors in vivo. Our findings provided the rationale for our initiation of Phase I and Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with hydroxychloroquine (HCQ) in PDAC. We have extended these findings and determined that the combined inhibition of KRASG12C or KRASG12D and autophagy was effective in KRAS-mutant cancers. Our ongoing studies are centered on developing additional combinations for targeting metabolic resistance mechanisms to KRAS or ERK MAPK targeted therapies. First, we performed a CRISPR/Cas-9 mediated genetic loss-of-function screen in the presence of CQ to determine additional sensitizers to autophagy inhibition. Top sensitizers included multiple facilitators of the DNA damage response, mTOR pathway components, and genes involved in the upstream regulation of the autophagy pathway. Additionally, we identified PIKfyve, a lipid kinase critical for the recycling dynamics of lysosomes, as an essential autophagy-related gene in PDAC cells. PIKfyve inhibition with apilimod resulted in a potent reduction of autophagic flux and growth. Importantly, PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the RAS ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. We conclude that concurrent inhibition of RAS and PIKfyve is a synergistic, cytotoxic combination that may represent a novel therapeutic strategy for PDAC. In a parallel line of investigation, we determined that autophagic signaling is temporally regulated following RAS pathway inhibition, peaking at 48-72 hours, and returning to basal levels by 7 days. Interestingly, we found that as autophagic upregulation subsides, another nutrient scavenging pathway, macropinocytosis, increases. We hypothesize that further dissection of the dynamic regulation of autophagy and macropinocytosis will improve current anti-nutrient scavenging treatment strategies. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. Citation Format: Jonathan M. DeLiberty, Ryan Robb, Mallory K. Roach, Sarah E. Ackermann, Runying Yang, Noah L. Pieper, Khalilah E. Taylor, Scott Bang, Elisa Baldelli, Emanuel F. Petricoin III, John P. Morris IV, Clint A. Stalnecker, Kirsten L. Bryant. Elucidation of metabolic resistance mechanisms to RAS inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-06.

Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression.

This study focuses on the role of lysosomal trafficking in prostate cancer, given the essential role of lysosomes in cellular homoeostasis. Lysosomal motility was evaluated using confocal laser scanning microscopy of LAMP-1-transfected prostate cells and spot-tracking analysis. Expression of lysosomal trafficking machinery was evaluated in patient cohort databases and through immunohistochemistry on tumour samples. The roles of vesicular trafficking machinery were evaluated through over-expression and siRNA. The effects of R1881 treatment on lysosome vesicular trafficking was evaluated by RNA sequencing, protein quantification and fixed- and live-cell microscopy. Altered regulation of lysosomal trafficking genes/proteins was observed in prostate cancer tissue, with significant correlations for co-expression of vesicular trafficking machinery in Gleason patterns. The expression of trafficking machinery was associated with poorer patient outcomes. R1881 treatment induced changes in lysosomal distribution, number, and expression of lysosomal vesicular trafficking machinery in hormone-sensitive prostate cancer cells. Manipulation of genes involved in lysosomal trafficking events induced changes in lysosome positioning and cell phenotype, as well as differential effects on cell migration, in non-malignant and prostate cancer cells. These findings provide novel insights into the altered regulation and functional impact of lysosomal vesicular trafficking in prostate cancer pathogenesis.

Long-term tracking of lysosomal dynamics with highly stable fluorescent probe

Monitoring lysosomal dynamics in real-time, especially in vivo, poses significant challenges due to the complex and dynamic nature of cellular environments. It is extremely important to construct fluorescent probes with high stability for imaging lysosomes to minimize interference from other cellular components, in order to ensure prolonged imaging. A fluorescent probe (PDB) has been proposed for targeting lysosomes, which was less affected to changes in the cellular microenvironment (such as pH, viscosity and polarity). PDB can be easily prepared by 4′-piperazinoacetophenone and 2-(4-diethylamino)-2-hydroxybenzoyl) benzoicacid, containing a piperazine group for labeling and imaging lysosomes and the high pKa value (∼9.35) allowed PDB to efficiently track lysosomes. The emission wavelength of PDB in aqueous solution was 634 nm (λex = 572 nm, Фf = 0.11). The dynamic process of lysosome induced by starvation and rapamycin was successfully explored by fluorescence imaging. Compared with the commercially available Lyso-Tracker green, the high photostability fluorescent probe can ensure 3D high-fidelity tracking and resist photobleaching. Therefore, PDB, unaffected by the cell microenvironment, successfully achieved long-term tracking of lysosomal movement, even enabling imaging in tumor-bearing mice over 11 days. The strong fluorescence signal, high stability, and long-term tracking capability indicate that PDB has tremendous potential in monitoring biological processes.

Pressure sensing of lysosomes enables control of TFEB responses in macrophages.

Polymers are endocytosed and hydrolysed by lysosomal enzymes to generate transportable solutes. While the transport of diverse organic solutes across the plasma membrane is well studied, their necessary ongoing efflux from the endocytic fluid into the cytosol is poorly appreciated by comparison. Myeloid cells that employ specialized types of endocytosis, that is, phagocytosis and macropinocytosis, are highly dependent on such transport pathways to prevent the build-up of hydrostatic pressure that otherwise offsets lysosomal dynamics including vesiculation, tubulation and fission. Without undergoing rupture, we found that lysosomes incurring this pressure owing to defects in solute efflux, are unable to retain luminal Na+, which collapses its gradient with the cytosol. This cation 'leak' is mediated by pressure-sensitive channels resident to lysosomes and leads to the inhibition of mTORC1, which is normally activated by Na+-coupled amino acid transporters driven by the Na+ gradient. As a consequence, the transcription factors TFEB/TFE3 are made active in macrophages with distended lysosomes. In addition to their role in lysosomal biogenesis, TFEB/TFE3 activation causes the release of MCP-1/CCL2. In catabolically stressed tissues, defects in efflux of solutes from the endocytic pathway leads to increased monocyte recruitment. Here we propose that macrophages respond to a pressure-sensing pathway on lysosomes to orchestrate lysosomal biogenesis as well as myeloid cell recruitment.

Lysosome dynamics during human endometrial stromal cells decidualization: effect of para-nonylphenol.

During the process of decidualization, the stromal cells of the endometrium change dynamically to create a favorable environment for embryo implantation. Lysosome activity has often been associated with physiological changes in the endometrium during the pre-implantation period and early pregnancy. In this study, the effect of para-nonylphenol (p-NP), an endocrine disruptor, on human immortalized endometrial stromal cells (tHESCs) was investigated. After exposure to p-NP (1 nM and 1 pM), the cells were examined for the decidualization markers Connexin-43, insulin like growth factor binding protein 1 (IGFBP1) and Prolactin. In addition, the effect of p-NP on lysosome biogenesis and exocytosis was investigated by examining the expression and localization of the transcription factor EB (TFEB) and that of the lysosomal-associated membrane protein 1 (LAMP-1). Finally, we evaluated the effect of p-NP on ECM remodeling using a fibronectin assay. Our results showed that p-NP reduced the expression of Prolactin protein, increased the nuclear localization of TFEB, and induced the increase and translocation of the lysosomal protein LAMP-1 to the membrane of tHESCs. The data indicate an impairment of decidualization and suggest an increase in lysosomal biogenesis and exocytosis, which is supported by the higher release of active cathepsin D by tHESCs. Given the importance of cathepsins in the processing and degradation of the ECM during trophoblast invasiveness and migration into the decidua, our results appear to be clear evidence of the negative effects of p-NP on endometrial processes that are fundamental to reproductive success and the establishment of pregnancy.

Age-related driving mechanisms of retinal diseases and neuroprotection by transcription factor EB-targeted therapy.

Retinal aging has been recognized as a significant risk factor for various retinal disorders, including diabetic retinopathy, age-related macular degeneration, and glaucoma, following a growing understanding of the molecular underpinnings of their development. This comprehensive review explores the mechanisms of retinal aging and investigates potential neuroprotective approaches, focusing on the activation of transcription factor EB. Recent meta-analyses have demonstrated promising outcomes of transcription factor EB-targeted strategies, such as exercise, calorie restriction, rapamycin, and metformin, in patients and animal models of these common retinal diseases. The review critically assesses the role of transcription factor EB in retinal biology during aging, its neuroprotective effects, and its therapeutic potential for retinal disorders. The impact of transcription factor EB on retinal aging is cell-specific, influencing metabolic reprogramming and energy homeostasis in retinal neurons through the regulation of mitochondrial quality control and nutrient-sensing pathways. In vascular endothelial cells, transcription factor EB controls important processes, including endothelial cell proliferation, endothelial tube formation, and nitric oxide levels, thereby influencing the inner blood-retinal barrier, angiogenesis, and retinal microvasculature. Additionally, transcription factor EB affects vascular smooth muscle cells, inhibiting vascular calcification and atherogenesis. In retinal pigment epithelial cells, transcription factor EB modulates functions such as autophagy, lysosomal dynamics, and clearance of the aging pigment lipofuscin, thereby promoting photoreceptor survival and regulating vascular endothelial growth factor A expression involved in neovascularization. These cell-specific functions of transcription factor EB significantly impact retinal aging mechanisms encompassing proteostasis, neuronal synapse plasticity, energy metabolism, microvasculature, and inflammation, ultimately offering protection against retinal aging and diseases. The review emphasizes transcription factor EB as a potential therapeutic target for retinal diseases. Therefore, it is imperative to obtain well-controlled direct experimental evidence to confirm the efficacy of transcription factor EB modulation in retinal diseases while minimizing its risk of adverse effects.

EIF2α-mediated integrated stress response links multiple intracellular signaling pathways to reprogram vascular smooth muscle cell fate in carotid artery plaque

BackgroundCarotid arterial atherosclerotic stenosis is a well-recognized pathological basis of ischemic stroke; however, its underlying molecular mechanisms remain unknown. Vascular smooth muscle cells (VSMCs) play fundamental roles in the initiation and progression of atherosclerosis. Organelle dynamics have been reported to affect atherosclerosis development. However, the association between organelle dynamics and various cellular stresses in atherosclerotic progression remain ambiguous. MethodsIn this study, we conducted transcriptomics and bioinformatics analyses of stable and vulnerable carotid plaques. Primary VSMCs were isolated from carotid plaques and subjected to histopathological staining to determine their expression profiles. Endoplasmic reticulum (ER), mitochondria, and lysosome dynamics were observed in primary VSMCs and VSMC cell lines using live-cell imaging. Moreover, the mechanisms underlying disordered organelle dynamics were investigated using comprehensive biological approaches. ResultsER whorls, a representative structural change under ER stress, are prominent dynamic reconstructions of VSMCs between vulnerable and stable plaques, followed by fragmented mitochondria and enlarged lysosomes, suggesting mitochondrial stress and lysosomal defects, respectively. Induction of mitochondrial stress alleviated ER stress and autophagy in an eukaryotic translation initiation factor (eIF)-2α-dependent manner. Furthermore, the effects of eIF2α on ER stress, mitochondrial stress, and lysosomal defects were validated using clinical samples. ConclusionOur results indicate that morphological and functional changes in VSMC organelles, especially in ER whorls, can be used as reliable biomarkers for atherosclerotic progression. Moreover, eIF2α plays an important role in integrating multiple stress-signaling pathways to determine the behavior and fate of VSMCs.

VPS13C regulates phospho-Rab10-mediated lysosomal function in human dopaminergic neurons.

Loss-of-function mutations in VPS13C are linked to early-onset Parkinson's disease (PD). While VPS13C has been previously studied in non-neuronal cells, the neuronal role of VPS13C in disease-relevant human dopaminergic neurons has not been elucidated. Using live-cell microscopy, we investigated the role of VPS13C in regulating lysosomal dynamics and function in human iPSC-derived dopaminergic neurons. Loss of VPS13C in dopaminergic neurons disrupts lysosomal morphology and dynamics with increased inter-lysosomal contacts, leading to impaired lysosomal motility and cellular distribution, as well as defective lysosomal hydrolytic activity and acidification. We identified Rab10 as a phospho-dependent interactor of VPS13C on lysosomes and observed a decreased phospho-Rab10-mediated lysosomal stress response upon loss of VPS13C. These findings highlight an important role of VPS13C in regulating lysosomal homeostasis in human dopaminergic neurons and suggest that disruptions in Rab10-mediated lysosomal stress response contribute to disease pathogenesis in VPS13C-linked PD.

Intrinsic Burst-Blinking Nanographenes for Super-Resolution Bioimaging.

Single-molecule localization microscopy (SMLM) is a powerful technique to achieve super-resolution imaging beyond the diffraction limit. Although various types of blinking fluorophores are currently considered for SMLM, intrinsic blinking fluorophores remain rare at the single-molecule level. Here, we report the synthesis of nanographene-based intrinsic burst-blinking fluorophores for highly versatile SMLM. We image amyloid fibrils in air and in various pH solutions without any additive and lysosome dynamics in live mammalian cells under physiological conditions. In addition, the single-molecule labeling of nascent proteins in primary sensory neurons was achieved with azide-functionalized nanographenes via click chemistry. SMLM imaging reveals higher local translation at axonal branching with unprecedented detail, while the size of translation foci remained similar throughout the entire network. These various results demonstrate the potential of nanographene-based fluorophores to drastically expand the applicability of super-resolution imaging.

Fluorogenic Rhodamine Probes with Pyrrole Substitution Enables STED and Lifetime Imaging of Lysosomes in Live Cells.

Fluorogenic dyes with high brightness, large turn-on ratios, excellent photostability, favorable specificity, low cytotoxicity, and high membrane permeability are essential for high-resolution fluorescence imaging in live cells. In this study, we endowed these desirable properties to a rhodamine derivative by simply replacing the N, N-diethyl group with a pyrrole substituent. The resulting dye, Rh-NH, exhibited doubled Stokes shifts (54 nm) and a red-shift of more than 50 nm in fluorescence spectra compared to Rhodamine B. Rh-NH preferentially exists in a non-emissive but highly permeable spirolactone form. Upon binding to lysosomes, the collective effects of low pH, low polarity, and high viscosity endow Rh-NH with significant fluorescence turn-on, making it a suitable candidate for wash-free, high-contrast lysosome tracking. Consequently, Rh-NH enabled us to successfully explore stimulated emission depletion (STED) super-resolution imaging of lysosome dynamics, as well as fluorescence lifetime imaging of lysosomes in live cells.

Abstract C047: Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. We and others recently demonstrated that inhibition of KRAS signaling through targeting the RAF-MEK-ERK kinase cascade resulted in further reliance on autophagy. We found that targeting this increased reliance on autophagy with the autophagy inhibitor hydroxychloroquine (HCQ)/chloroquine (CQ) together with MEK or ERK inhibition (MEKi, ERKi) synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, a limitation of this approach is that HCQ/CQ are not specific or potent autophagy inhibitors. Thus, we sought to identify a more efficacious autophagy inhibition strategy. To this end, we performed a CRISPR-Cas9 mediated genetic loss-of-function screen in PDAC cells to identify other targetable mediators of autophagy. We identified PIKfyve, a lipid kinase critical for the recycling dynamics of lysosomes, as an essential autophagy-related gene in PDAC cells. PIKfyve inhibition by the clinical candidate inhibitor apilimod resulted in potent reduction of autophagic flux and growth. Additionally, PIKfyve inhibition induced intracellular vacuolization. These PIKfyve-regulated vacuoles stained positive for the lysosomal marker, LAMP1, and exhibited reduced acidity and impaired cargo degradation. Importantly, PIKfyve inhibition prevented the increased autophagic flux we observed when we inhibited MEK with the clinical stage MEKi, mirdametinib. As a result, co-targeting MEK and PIKfyve led to synergistic impairment of PDAC cell proliferation. Similar results were observed following dual inhibition of KRAS and PIKfyve. We found the synergistic growth inhibition was due in part to a substantial induction of apoptosis unique to combination treatment. We then tested the combination of MEKi and PIKfyvei in a panel of patient derived PDAC organoids and observed a robust synergistic reduction in viability, suggesting this may be an efficacious therapeutic strategy for PDAC treatment. Future work includes in vivo studies, currently underway, to determine the efficacy of single agent PIKfyve inhibition, as well as efficacy studies assessing combined MEKi and PIKfyvei in orthotopic mouse models of PDAC. Taken together, these findings implicate PIKfyve as an effective anti-autophagy target when paired with RAS or ERK-MAPK pathway inhibition in pre-clinical models of pancreatic cancer. Citation Format: Jonathan M. DeLiberty, Mallory K. Roach, Noah L. Pieper, Kristina Drizyte-Miller, Elyse G. Schechter, Runying Yang, Channing J. Der, Adrienne D. Cox, Clint A. Stalnecker, Kirsten L. Bryant. Improving the efficacy of dual ERK-MAPK and autophagy inhibition as a therapeutic strategy for pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C047.

Unveiling autophagy and aging through time-resolved imaging of lysosomal polarity with a delayed fluorescent emitter.

Detecting the lysosomal microenvironmental changes like viscosity, pH, and polarity during their dynamic interorganelle interactions remains an intriguing area that facilitates the elucidation of cellular homeostasis. The subtle variation of physiological conditions can be assessed by deciphering the lysosomal microenvironments during lysosome-organelle interactions, closely related to autophagic pathways leading to various cellular disorders. Herein, we shed light on the dynamic lysosomal polarity in live cells and a multicellular model organism, Caenorhabditis elegans (C. elegans), through time-resolved imaging employing a thermally activated delayed fluorescent probe, DC-Lyso. The highly photostable and cytocompatible DC-Lyso rapidly labels the lysosomes (within 1 min of incubation) and exhibits red luminescence and polarity-sensitive long lifetime under the cellular environment. The distinct variation in the fluorescence lifetime of DC-Lyso suggests an increase in local polarity during the lysosomal dynamics and interorganelle interactions, including lipophagy and mitophagy. The lifetime imaging analysis reveals increasing lysosomal polarity as an indicator for probing the successive development of C. elegans during aging. The in vivo microsecond timescale imaging of various cancerous cell lines and C. elegans, as presented here, therefore, expands the scope of delayed fluorescent emitters for unveiling complex biological processes.

Visualizing Lipophagy as a New Mechanism of the Synthesis of Sex Steroids in Human Ovary and Testis Using Immunofluorescence Staining Method.

Immunofluorescence, a transformative tool in cellular biology, is employed to dissect the intricate mechanisms of cholesterol trafficking in human reproductive tissues. Autophagy, a key player in cellular homeostasis, particularly lipophagy, emerges as a free cholesterol source for steroidogenesis. In this chapter, we describe a comprehensive immunofluorescence staining protocol, with details provided for the precise visualization of subcellular dynamics of mitochondria, lysosomes, and lipid droplets in ex vivo testicular tissue and primary luteal granulosa cell culture models, pivotal components in sex steroid biosynthesis. Here, we detail the culture, treatment, and immunofluorescence protocols, providing a comprehensive guide for researchers. The provided immunofluorescence toolkit serves as a valuable resource for researchers, paving way for advancements in human reproductive health to investigate the intricate interplay between autophagy, lipophagy, and cholesterol trafficking.

A zinc metal complex as an NIR emissive probe for real-time dynamics and in vivo embryogenic evolution of lysosomes using super-resolution microscopy.

Zinc (Zn) based fluorescent metal complexes have gained increasing attention due to their non-toxicity and high brightness with marked fluorescence quantum yield (QY). However, they have rarely been employed in super-resolution microscopy (SRM) to study live cells and in vivo dynamics of lysosomes. Here, we present an NIR emissive highly photostable Zn-complex as a multifaceted fluorescent probe for the long-term dynamical distribution of lysosomes in various cancerous and non-cancerous cells in live condition and in vivo embryogenic evolution in Caenorhabditis elegans (C. elegans). Apart from the normal fission, fusion, and kiss & run, the motility and the exact location of lysosomes at each point were mapped precisely. A notable difference in the lysosomal motility in the peripheral region between cancerous and non-cancerous cells was distinctly observed. This is attributed to the difference in viscosity of the cytoplasmic environment. On the other hand, along with the super-resolved structure of the smallest size lysosome (∼77 nm) in live C. elegans, the complete in vivo embryogenic evolution of lysosomes and lysosome-related organelles (LROs) was captured. We were able to capture the images of lysosomes and LROs at different stages of C. elegans, starting from a single cell and extending to a fully matured adult animal.

Biallelic BORCS8 variants cause an infantile-onset neurodegenerative disorder with altered lysosome dynamics.

BLOC-one-related complex (BORC) is a multiprotein complex composed of eight subunits named BORCS1-8. BORC associates with the cytosolic face of lysosomes, where it sequentially recruits the small GTPase ARL8 and kinesin-1 and -3 microtubule motors to promote anterograde transport of lysosomes toward the peripheral cytoplasm in non-neuronal cells and the distal axon in neurons. The physiological and pathological importance of BORC in humans, however, remains to be determined. Here, we report the identification of compound heterozygous variants [missense c.85T>C (p.Ser29Pro) and frameshift c.71-75dupTGGCC (p.Asn26Trpfs*51)] and homozygous variants [missense c.196A>C (p.Thr66Pro) and c.124T>C (p.Ser42Pro)] in BORCS8 in five children with a severe early-infantile neurodegenerative disorder from three unrelated families. The children exhibit global developmental delay, severe-to-profound intellectual disability, hypotonia, limb spasticity, muscle wasting, dysmorphic facies, optic atrophy, leuko-axonopathy with hypomyelination, and neurodegenerative features with prevalent supratentorial involvement. Cellular studies using a heterologous transfection system show that the BORCS8 missense variants p.Ser29Pro, p.Ser42Pro and p.Thr66Pro are expressed at normal levels but exhibit reduced assembly with other BORC subunits and reduced ability to drive lysosome distribution toward the cell periphery. The BORCS8 frameshift variant p.Asn26Trpfs*51, on the other hand, is expressed at lower levels and is completely incapable of assembling with other BORC subunits and promoting lysosome distribution toward the cell periphery. Therefore, all the BORCS8 variants are partial or total loss-of-function alleles and are thus likely pathogenic. Knockout of the orthologous borcs8 in zebrafish causes decreased brain and eye size, neuromuscular anomalies and impaired locomotion, recapitulating some of the key traits of the human disease. These findings thus identify BORCS8 as a novel genetic locus for an early-infantile neurodegenerative disorder and highlight the critical importance of BORC and lysosome dynamics for the development and function of the central nervous system.

Lysosomal dynamics regulate mammalian cortical neurogenesis

Mammalian neocortex formation follows a stereotypical pattern wherein the self-renew and differentiation of neural stem cells are coordinated with diverse organelle dynamics. However, the role of lysosomes in brain development has long been overlooked. Here, we demonstrate the highly dynamic lysosomal quantities, types, and localizations in developing brain. We observed asymmetric endolysosome inheritance during radial glial cell (RGC) division and the increased autolysosomes within intermediate progenitor cells (IPs) and newborn neurons. Disruption of lysosomal function shortens the S phase of the cell cycle and promotes RGC differentiation. Mechanistically, we revealed a post-transcriptional regulation governing ribosome homeostasis and cell-cycle progression through differential lysosomal activity modulation. In the human forebrain organoid, lysosomal dynamics are conserved; specifically, during the mitosis of outer subventricular zone RGCs (oRGs), lysosomes are inherited by the progeny without basal process. Together, our results identify the critical role of lysosomal dynamics in regulating mouse and human brain development.

A new role for phosphoinositides in regulating mitochondrial dynamics

Phosphoinositides are a minor group of membrane-associated phospholipids that are transiently generated on the cytoplasmic leaflet of many organelle membranes and the plasma membrane. There are seven functionally distinct phosphoinositides, each derived via the reversible phosphorylation of phosphatidylinositol in various combinations on the inositol ring. Their generation and termination is tightly regulated by phosphatidylinositol-kinases and –phosphatases. These enzymes can function together in an integrated and coordinated manner, whereby the phosphoinositide product of one enzyme may subsequently serve as a substrate for another to generate a different phosphoinositide species. This regulatory mechanism not only enables the transient generation of phosphoinositides on membranes, but also more complex sequential or bidirectional conversion pathways, and phosphoinositides can also be transferred between organelles via membrane contacts. It is this capacity to fine-tune phosphoinositide signals that makes them ideal regulators of membrane organization and dynamics, through their recruitment of signalling, membrane altering and lipid transfer proteins. Research spanning several decades has provided extensive evidence that phosphoinositides are major gatekeepers of membrane organization, with roles in endocytosis, exocytosis, autophagy, lysosome dynamics, vesicular transport and secretion, cilia, inter-organelle membrane contact, endosome maturation and nuclear function. By contrast, there has been remarkably little known about the role of phosphoinositides at mitochondria – an enigmatic and major knowledge gap, with challenges in reliably detecting phosphoinositides at this site.Here we review recent significant breakthroughs in understanding the role of phosphoinositides in regulating mitochondrial dynamics and metabolic function.