Embedding of HIV Egress within Cortical F-Actin

F-Actin remodeling is important for the spread of HIV via cell–cell contacts; however, the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell–cell contact, provided Cdc42 and IQGAP1 were present. From these observations, we conclude that a proportion out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, the maturation of cell–cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.

Afadin couples RAS GTPases to the polarity rheostat Scribble

AFDN/Afadin is required for establishment and maintenance of cell-cell contacts and is a unique effector of RAS GTPases. The biological consequences of RAS signalling to AFDN are unknown. Here, we use proximity-based proteomics to generate an interaction map for the long and short isoforms of AFDN, identifying the polarity protein SCRIB/Scribble as the top hit. We reveal that the first PDZ domain of SCRIB and the AFDN FHA domain mediate a direct but non-canonical interaction between these important adhesion and polarity proteins. Further, the dual RA domains of AFDN have broad specificity for RAS and RAP GTPases, and KRAS co-localizes with and promotes AFDN-SCRIB complex formation. Knockout of AFDN or SCRIB in MCF7 epithelial cells disrupts MAPK and PI3K activation and inhibits cell motility in a growth factor-dependent manner. These data have important implications for understanding why cells with activated RAS have reduced cell contacts and polarity defects, and finally begin to characterize AFDN as a RAS effector.

Zonula occludens 2 and Cell-Cell Contacts Are Required for Normal Nuclear Shape in Epithelia

MAGUK protein ZO-2 is present at tight junctions (TJs) and nuclei. In MDCK ZO-2 knockdown (KD) cells, nuclei exhibit an irregular shape with lobules and indentations. This condition correlates with an increase in DNA double strand breaks, however cells are not senescent and instead become resistant to UV-induced senescence. The irregular nuclear shape is also observed in isolated cells and in those without TJs, due to the lack of extracellular calcium. The aberrant nuclear shape of ZO-2 KD cells is not accompanied by a reduced expression of lamins A/C and B and lamin B receptors. Instead, it involves a decrease in constitutive and facultative heterochromatin, and microtubule instability that is restored with docetaxel. ZO-2 KD cells over-express SUN-1 that crosses the inner nuclear membrane and connects the nucleoskeleton of lamin A to nesprins, which traverse the outer nuclear membrane. Nesprins-3 and -4 that indirectly bind on their cytoplasmic face to vimentin and microtubules, respectively, are also over-expressed in ZO-2 KD cells, whereas vimentin is depleted. SUN-1 and lamin B1 co-immunoprecipitate with ZO-2, and SUN-1 associates to ZO-2 in a pull-down assay. Our results suggest that ZO-2 forms a complex with SUN-1 and lamin B1 at the inner nuclear membrane, and that ZO-2 and cell–cell contacts are required for a normal nuclear shape.

Ultrastructural changes in the microvessels and perivascular space in cerebral infarction in the experiment

Background. Ischemic stroke is the second most common cause of death after coronary heart disease and the most common cause of disability worldwide. Much of the recent basic research on stroke is concerned with the mechanisms underlying the dysfunction and adaptation of the neurovascular block, which includes the blood-brain barrier structures, microglia, neurons, and the extracellular matrix of the basement membrane. Isolated studies of recent years have been devoted to the issues of morphology and in particular the ultrastructure of the brain in ischemic injury. Meanwhile, only morphological studies can reveal the peculiarities of the response of cellular structures to the influence of various adverse factors. Objective – to investigate ultrastructural changes in the vessels of the brain and perivascular space in experimental ischemic heart attack. Methods. Experimental cerebral infarction was reproduced on 15 white Wistar rats by injection of a suspension of barium sulfate in sterile saline in a ratio of 1: 3 in the amount of 0.1 -0.3 ml. Three animals formed a control group. The material was collected in terms of: up to 3, 9, 12 days and more than 12 days from the beginning of the experimental action, followed by standard processing of the material for electron microscopy. Results. In the early stages of ischemic brain damage perivascular edema, destructive changes of capillaries with destruction of basement membranes are registered. Some microvessels undergo irreversible changes with deformation of the vascular lumen, pyknosis and lysis of endothelial nuclei, destruction and vacuolation of cytoplasmic structures, microvacuolation and edema of mitochondria with partial destruction of cristae and enlightenment of the mitochondrial matrix. In the endothelium with signs of coagulation processes in the cytoplasm and nucleus, changes in cell contacts were observed. Structural changes of vessels are combined with changes of perivascular processes of astrocytes. On days 9 and 12, the structure of the endothelium, perivascular astrocytes, and intercellular contacts are restored. Hyperplasia of intracytoplasmic structures, increase in mitochondria and length of cytoplasmic network are noted. In the cells of the perivascular environment and in the cytoplasm of pericytes a significant number of phagolysosomes is detected, in the long term in the perifocal areas of irreversible ischemic changes around the vessels is reparative astrogliosis. Conclusion. Ultrastructural changes of the microcirculatory part in the perifocal areas of ischemic lesions within 3 days are characterized by perivascular edema and destructive changes in the endothelium of capillaries and pericytes, damage to basement membranes, changes in cell contacts. After 9-12 days in the endothelium, the processes of intracellular regeneration increase, the ultrastructure of intercellular contacts is restored. A significant number of phagolysosomes is registered in the cells of the perivascular environment and in the cytoplasm of pericytes, and reparative astrogliosis is detected in the perifocal areas of irreversible ischemic changes around the vessels.

Ferroptosis Meets Cell–Cell Contacts

Ferroptosis is a regulated form of cell death characterized by iron dependency and increased lipid peroxidation. Initially assumed to be selectively induced in tumour cells, there is increasing evidence that ferroptosis plays an important role in pathophysiology and numerous cell types and tissues. Deregulated ferroptosis has been linked to human diseases, such as neurodegenerative diseases, cardiovascular disorders, and cancer. Along these lines, ferroptosis is a promising pathway to overcoming therapy resistance of cancer cells. It is therefore of utmost importance to understand the cellular signalling pathways and the molecular mechanisms underlying ferroptosis regulation, including context-specific effects mediated by the neighbouring cells through cell–cell contacts. Here, we give an overview on the molecular events and machinery linked to ferroptosis induction and commitment. We further summarize and discuss current knowledge about the role of cell–cell contacts, which differ in ferroptosis regulation between normal somatic cells and cancer cells. We present emerging concepts on the underlying mechanisms, address open questions, and discuss the possible impact of cell–cell contacts on exploiting ferroptosis in cancer therapy.

Human DLG1 and SCRIB Are Distinctly Regulated Independently of HPV-16 during the Progression of Oropharyngeal Squamous Cell Carcinomas: A Preliminary Analysis

The major causative agents of head and neck squamous cell carcinomas (HNSCCs) are either environmental factors, such as tobacco and alcohol consumption, or infection with oncogenic human papillomaviruses (HPVs). An important aspect of HPV-induced oncogenesis is the targeting by the E6 oncoprotein of PDZ domain-containing substrates for proteasomal destruction. Tumor suppressors DLG1 and SCRIB are two of the principal PDZ domain-containing E6 targets. Both have been shown to play critical roles in the regulation of cell growth and polarity and in maintaining the structural integrity of the epithelia. We investigated how modifications in the cellular localization and protein expression of DLG1 and SCRIB in HPV16-positive and HPV-negative histologic oropharyngeal squamous cell carcinomas (OPSCC) might reflect disease progression. HPV presence was determined by p16 staining and HPV genotyping. Whilst DLG1 expression levels did not differ markedly between HPV-negative and HPV16-positive OPSCCs, it appeared to be relocated from cell–cell contacts to the cytoplasm in most samples, regardless of HPV16 positivity. This indicates that alterations in DLG1 distribution could contribute to malignant progression in OPSCCs. Interestingly, SCRIB was also relocated from cell–cell contacts to the cytoplasm in the tumor samples in comparison with normal tissue, regardless of HPV16 status, but in addition there was an obvious reduction in SCRIB expression in higher grade tumors. Strikingly, loss of SCRIB was even more pronounced in HPV16-positive OPSCCs. These alterations in SCRIB levels may contribute to transformation and loss of tissue architecture in the process of carcinogenesis and could potentially serve as markers in the development of OPSCCs.