ARTICLE | doi:10.20944/preprints201810.0521.v1
Subject: Medicine & Pharmacology, Gastroenterology Keywords: alcohol-induced Golgi disorganization; Golgi recovery; giantin; hepatic proteins; ethanol withdrawal
Online: 23 October 2018 (06:10:04 CEST)
Background: In hepatocytes and alcohol-metabolizing cultured cells, Golgi undergoes ethanol (EtOH)-induced disorganization. Periniclear and organized Golgi is important in liver homeostasis, but how the Golgi remains intact is unknown. Work from our laboratories showed that EtOH-altered cellular function could be reversed after alcohol removal; we wanted to determine whether this recovery would apply to Golgi. Methods: We used alcohol-metabolizing HepG2 (VA-13) cells (cultured with or without EtOH for 72 h) and rat hepatocytes (control and EtOH-fed (Lieber-DeCarli diet). For recovery, EtOH was removed and replenished with control medium (48 hours for VA-13 cells) or control diet (10 days for rats). Results: EtOH-induced Golgi disassembly was associated with de-dimerization of the largest Golgi matrix protein giantin, along with impaired transport of selected hepatic proteins. After recovery from EtOH, Golgi regained their compact structure, and alterations in giantin and protein transport were restored. In VA-13 cells, when we knocked down giantin, Rab6a GTPase or non-muscle Myosin IIB, minimal changes were observed in control conditions, but post-EtOH recovery was impaired. Conclusions: These data provide a link between Golgi organization and plasma membrane protein expression and identify several proteins whose expression is important to maintain Golgi structure during the recovery phase after EtOH administration.
REVIEW | doi:10.20944/preprints202007.0601.v1
Subject: Biology, Other Keywords: traffic; endocytosis; sorting; Aspergillus nidulans; UapA; Golgi; fungi
Online: 25 July 2020 (11:00:02 CEST)
Eukaryotic plasma membrane (PM) transporters face critical challenges that are not widely present in prokaryotes. The two most important issues are proper subcellular traffic and targeting to the PM, and regulated endocytosis in response to physiological, developmental or stress signals. Sorting of transporters from their site of synthesis, the Endoplasmic Reticulum (ER), to the PM has been long thought, but not formally shown, to occur via the conventional Golgi-dependent vesicular secretory pathway. Endocytosis of specific eukaryotic transporters has been studied more systematically and shown to involve ubiquitination, internalization, and sorting to early endosomes, followed by turnover in the MVB/lysosomes/vacuole system. In specific cases internalized transporters have been shown to recycle back to the PM. However, the mechanisms of transporter forward trafficking and turnover have been overturned recently through systematic work in the model fungus Aspergillus nidulans. In this review we present evidence that shows that transporter traffic to the PM takes place through Golgi-bypass and transporter endocytosis operates via a mechanism that is distinct from that of recycling membrane cargoes essential for fungal growth. We discuss these findings in relation to adaptation to challenges imposed by cell polarity in fungi as well as in other eukaryotes and provide a rationale why transporters and possibly other housekeeping membrane proteins ‘avoid’ routes of polar trafficking.
Subject: Medicine & Pharmacology, Pathology & Pathobiology Keywords: Epilepsy; Brain damage; Golgi determinations; Ramon y Cajal: axons
Online: 27 March 2020 (12:35:20 CET)
As a medical student (Granada University Medical School, Spain), interested in Pediatrics, expended countless hours at the hospital pediatric facilities and got to know many of the children and their medical problems. A particular case, still vivid on my mind, awaken my scientific curiosity. One day, walking and talking with a seven years old child he unexpectedly felt down unconscious with multiple, incontrollable and erratic muscular contractions involving face, body and extremities and salivating. I was overwhelmed thinking it was the child’ last hour. At the time, my knowledge of epilepsy was nil. Following the seizures, the child was up, talking and walking with me as if nothing has happened and without any knowledge of the event. What could have caused the brain motor cortex to suddenly discharge that amount of altered activity causing generalized and erratic muscular contractions remains inexplicable. I migrated to USA, become a Pediatric (Developmental) Pathologist and Director of the Pediatrics Autopsy Service (1962-1999) at the Dartmouth-Hitchcock Medical Center, New Hampshire. I carried out countless postmortem studied of children brains, normal (unaltered) as well as those altered by hemorrhagic, hypoxic-ischemic and/or traumatic damage. With an NIH Fellowship, I spend one year (1967-68) at the Cajal Institute (Madrid, Spain) studying Cajal’ old Golgi preparations and learning about the method. Some of my Golgi studies of children’ brains have been published: The Human Brain. Prenatal Development and Structure, Springer, Heidelberg, Germany, 2012. The present monograph explores the developmental neuropathology of selected perinatal cortical injuries through their acute, subacute and chronic stages. Including: a) how an altered neuronal activity evolves in a damaged cortical region; b) how it moves through the cortex (epileptic auras); and c) how it reaches the motor cortex to be discharged as erratic and incontrollable muscular contractions. Understanding these processes should provide insights into the pathogenesis of epilepsy secondary to perinatal brain damage.
Subject: Life Sciences, Biophysics Keywords: cytosolic phospholipase A2; electron microscopy; C2 domain; Golgi; membrane bending; membrane curvature; oligomerization
Online: 12 March 2020 (04:10:52 CET)
Group IV phospholipase A2α (cPLA2α) regulates the production of prostaglandins and leukotrienes via the formation of arachidonic acid from membrane phospholipids. The targeting and membrane binding of cPLA2α to the Golgi involves the N-terminal C2 domain whereas the catalytic domain produces arachidonic acid. Although most studies of cPLA2α concern its catalytic activity, it is also linked to homeostatic processes involving the generation of vesicles that traffic material from the Golgi to the plasma membrane. Here we investigate how membrane curvature influences the homeostatic role of cPLA2α in vesicular trafficking. The cPLA2α C2 domain is known to induce changes in positive membrane curvature, a process which is dependent on cPLA2α membrane penetration. We show that cPLA2α undergoes C2 domain-dependent oligomerization on membranes in vitro and in A549 cells. We found that the association of the cPLA2α C2 domain with membranes is limited to membranes with positive curvature, and enhanced C2 domain oligomerization was observed on vesicles ~50 nm in diameter. We demonstrated that the cPLA2α C2 domain generates cholesterol enriched Golgi-derived vesicles independently of cPLA2α catalytic activity. Our results therefore provide novel insight into the molecular forces that mediate C2 domain-dependent membrane localization in vitro and in cells.
ARTICLE | doi:10.20944/preprints201908.0027.v1
Subject: Biology, Plant Sciences Keywords: plant specific insert; aspartic proteinase; vacuolar sorting; unconventional trafficking; endoplasmic reticulum; Golgi, N-linked glycosylation
Online: 2 August 2019 (10:05:06 CEST)
In plant cells the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSIs domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlike its native glycosylated form, is able to bypass ER-to-Golgi blockage and accumulate in the vacuole. This is also true when the PSI domain is analyzed in the context of the full-length cardosin. Regardless of opening exciting research gaps, the results obtained so far need a more comprehensive study of the mechanisms behind this unconventional direct sorting to the vacuole.