Vitamin C may exert variable effects on viability and proliferation of HeLa cells exhibiting high and low chromosomal instability.

BACKGROUND: Chromosomal instability (CIN), defined as abnormality in chromosome structure or number, is the hallmark of malignancies. The role of vitamin C in cancer treatment is controversial and its effect on CIN is still an open field of research. In this work, we tried to study the effect of high-dose L-ascorbic acid (L-AA) on CIN-induced (CINhi = CIN high) and non-CIN-induced (CINlo = CIN low) cervical cancer cells. OBJECTIVES: The aim of this study was to explore the effect of high-dose L-AA on CIN in the cervical cancer cell line (HeLa) cells. MATERIAL AND METHODS: The HeLa cells (CINhi and CINlo) were treated with 2 doses (5 mM and 8 mM) of L-AA for 24 h and 48 h. They were then analyzed by micronucleus (MN) scoring, cell ploidy and flow cytometry, the latter regarding γH2AX expression. Cell viability was assessed by the methylthiazol tetrazolium (MTT) and Annexin V assays. RESULTS: Treatment of CINhi cells with L-AA led to a decrease in MN score (colchicine - 71.5 ±4.95, 67.5 ±0.71; L-AA (5 mM) - 49 ±7.07, 46.5 ±4.95; L-AA (8 mM) - 42 ±9.89, 41 ±1.41, at 24 h and 48 h, respectively; p < 0.05). Treatment of CINlo cells with L-AA resulted in increased MN score (5 mM - 45 ±7.07, 36 ±4.24; 8 mM - 34.5 ±4.95, 31 ±1.41, at 24 h and 48 h, respectively; control - 15.5 ±0.71, 12.5 ±0.71; p < 0.05) and reduction in cancer cell viability (control - 100%; L-AA (5 mM) - 76.32% ±28.73, 72.74% ±20.30; L-AA (8 mM) - 66.14% ±19.13, 66.99% ±19.99, at 24 h and 48 h, respectively; p < 0.05). The expression of γH2AX was high in both groups at 48 h (mean CINhi = 19.42%, CINlo = 21.14%; control = 1.19% and 1.58%, respectively) with the 8 mM dose of L-AA. CONCLUSIONS: L-ascorbic acid was found to have a differential effect on CINhi and CINlo HeLa cells, which may be due to differences in oxidation status of these 2 categories.

Salmonella exploits the host endolysosomal tethering factor HOPS complex to promote its intravacuolar replication.

Salmonella enterica serovar typhimurium extensively remodels the host late endocytic compartments to establish its vacuolar niche within the host cells conducive for its replication, also known as the Salmonella-containing vacuole (SCV). By maintaining a prolonged interaction with late endosomes and lysosomes of the host cells in the form of interconnected network of tubules (Salmonella-induced filaments or SIFs), Salmonella gains access to both membrane and fluid-phase cargo from these compartments. This is essential for maintaining SCV membrane integrity and for bacterial intravacuolar nutrition. Here, we have identified the multisubunit lysosomal tethering factor-HOPS (HOmotypic fusion and Protein Sorting) complex as a crucial host factor facilitating delivery of late endosomal and lysosomal content to SCVs, providing membrane for SIF formation, and nutrients for intravacuolar bacterial replication. Accordingly, depletion of HOPS subunits significantly reduced the bacterial load in non-phagocytic and phagocytic cells as well as in a mouse model of Salmonella infection. We found that Salmonella effector SifA in complex with its binding partner; SKIP, interacts with HOPS subunit Vps39 and mediates recruitment of this tethering factor to SCV compartments. The lysosomal small GTPase Arl8b that binds to, and promotes membrane localization of Vps41 (and other HOPS subunits) was also required for HOPS recruitment to SCVs and SIFs. Our findings suggest that Salmonella recruits the host late endosomal and lysosomal membrane fusion machinery to its vacuolar niche for access to host membrane and nutrients, ensuring its intracellular survival and replication.

The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes.

The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit-subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation.

Arf-like GTPase Arl8: Moving from the periphery to the center of lysosomal biology.

Lysosomes are dynamic organelles that not only mediate degradation of cellular substrates but also play critical roles in processes such as cholesterol homeostasis, plasma membrane repair, antigen presentation, and cell migration. The small GTPase Arl8, a member of Arf-like (Arl) family of proteins, has recently emerged as a crucial regulator of lysosome positioning and membrane trafficking toward lysosomes. Through interaction with its effector SKIP, the human Arl8 paralog (Arl8b) mediates kinesin-1 dependent motility of lysosomes on microtubule tracks toward the cell periphery. Arl8b-mediated kinesin-driven motility is also implicated in regulating lytic granule polarization in NK cells, lysosome tubulation in macrophages, cell spreading, and migration. Moreover, Arl8b regulates membrane traffic toward lysosomes by recruiting subunits of the HOPS complex, a multi-subunit tethering complex that mediates endo-lysosome fusion. Here we provide a brief review on this recently characterized lysosomal GTPase and summarize the studies focusing on its known functions in regulating lysosomal motility and delivery of endocytic cargo to the lysosomes. We also explore the role of human Arl8b and its orthologs upon infection by intracellular pathogens.