The actin cytoskeleton mediates a variety of essential biological functions in all eukaryotic cells. In addition to providing a structural framework around which cell shape and polarity are defined, its dynamic properties provide the driving force for cells to move and to divide. Understanding the biochemical mechanisms that control the organization of actin is thus a major goal of contemporary cell biology, with implications for health and disease. Members of the Rho family of small guanosine triphosphatases have emerged as key regulators of the actin cytoskeleton, and furthermore, through their interaction with multiple target proteins, they ensure coordinated control of other cellular activities such as gene transcription and adhesion.

/pdf/rho-gtpases-and-the-actin-cytoskeleton-zsfyn1yhxi.pdf

Rho GTPases and the Actin Cytoskeleton

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Signal transduction by the JNK group of MAP kinases.

Mitogen-activated protein (MAP) kinases comprise a family of ubiquitous proline-directed, protein-serine/threonine kinases, which participate in signal transduction pathways that control intracellular events including acute responses to hormones and major developmental changes in organisms. MAP kinases lie in protein kinase cascades. This review discusses the regulation and functions of mammalian MAP kinases. Nonenzymatic mechanisms that impact MAP kinase functions and findings from gene disruption studies are highlighted. Particular emphasis is on ERK1/2.

/pdf/mitogen-activated-protein-map-kinase-pathways-regulation-and-3nsauvepr2.pdf

Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions.

Cellular organelles in the exocytic and endocytic pathways have a distinctive spatial distribution and communicate through an elaborate system of vesiculo-tubular transport. Rab proteins and their effectors coordinate consecutive stages of transport, such as vesicle formation, vesicle and organelle motility, and tethering of vesicles to their target compartment. These molecules are highly compartmentalized in organelle membranes, making them excellent candidates for determining transport specificity and organelle identity.

/pdf/rab-proteins-as-membrane-organizers-4nbrkb2cqi.pdf

Rab proteins as membrane organizers

Glioblastoma Multiforme (GBM) is one of the most lethal human cancer types, with a 5-year survival rate of approximately 5%. A key reason for this is usually considered its poor accessibility to systemically administered drugs that only limitedly overcome the Blood Brain Barrier, ultimately causing the likely dismal appearance of recurrences. Here, we comment on our successful use, in GBM preclinical models, of novel thermogel based drug-delivery platforms for loco-regional treatment of tumor recurrences after primary surgery. The innovation as well as the pitfalls of our processes are outlined and discussed with an eye towards potential advancements in the realm of personalized medicine applications.

Aiming for the brain: a new thermogel-based drug delivery platform

Abstract Background In Neisseria meningitidis the HrpA/HrpB two-partner secretion system (TPS) was implicated in diverse functions including meningococcal competition, biofilm formation, adherence to epithelial cells, intracellular survival and vacuolar escape. These diverse functions could be attributed to distinct domains of secreted HrpA. Methods A yeast two-hybrid screening, in vitro pull-down assay and immunofluorescence microscopy experiments were used to investigate the interaction between HrpA and the dynein light-chain, Tctex-type 1 (DYNLT1). In silico modeling was used to analyze HrpA structure. Western blot analysis was used to investigate apoptotic and pyroptotic markers. Results The HrpA carboxy-terminal region acts as a manganese-dependent cell lysin, while the results of a yeast two-hybrid screening demonstrated that the HrpA middle region has the ability to bind the dynein light-chain, Tctex-type 1 (DYNLT1). This interaction was confirmed by in vitro pull-down assay and immunofluorescence microscopy experiments showing co-localization of N. meningitidis with DYNLT1 in infected epithelial cells. In silico modeling revealed that the HrpA-M interface interacting with the DYNLT1 has similarity with capsid proteins of neurotropic viruses that interact with the DYNLT1. Indeed, we found that HrpA plays a key role in infection of and meningococcal trafficking within neuronal cells, and is implicated in the modulation of the balance between apoptosis and pyroptosis. Conclusions Our findings revealed that N. meningitidis is able to effectively infect and survive in neuronal cells, and that this ability is dependent on HrpA, which establishes a direct protein–protein interaction with DYNLTI in these cells, suggesting that the HrpA interaction with dynein could be fundamental for N. meningitidis spreading inside the neurons. Moreover, we found that the balance between apoptotic and pyroptotic pathways is heavily affected by HrpA. 

https://jbiomedsci.biomedcentral.com/counter/pdf/10.1186/s12929-022-00829-8

HrpA anchors meningococci to the dynein motor and affects the balance between apoptosis and pyroptosis

During skeletal myogenesis, the zinc‐finger transcription factors SNAI1 and SNAI2, are expressed in proliferating myoblasts and regulate the transition to terminally differentiated myotubes while repressing pro‐differentiation genes. Here, we demonstrate that SNAI1 is upregulated in vivo during the early phase of muscle regeneration induced by bupivacaine injury. Using shRNA‐mediated gene silencing in C2C12 myoblasts and whole‐transcriptome microarray analysis, we identified a collection of genes belonging to the endoplasmic reticulum (ER) stress pathway whose expression, induced by myogenic differentiation, was upregulated in absence of SNAI1. Among these, key ER stress genes, such as Atf3, Ddit3/Chop, Hspa5/Bip, and Fgf21, a myokine involved in muscle differentiation, were strongly upregulated. Furthermore, by promoter mutant analysis and Chromatin immune precipitation assay, we demonstrated that SNAI1 represses Fgf21 and Atf3 in proliferating myoblasts by directly binding to multiple E boxes in their respective promoter regions. Together, these data describe a new regulatory mechanism of myogenic differentiation involving the direct repressive action of SNAI1 on ER stress and Fgf21 expression, ultimately contributing to maintaining the proliferative and undifferentiated state of myoblasts.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1096/fj.202200215R

SNAI1 is upregulated during muscle regeneration and represses FGF21 and ATF3 expression by directly binding their promoters

/pdf/hrpa-anchors-meningococci-to-the-dynein-motor-and-affects-3r4wsdu6.pdf

Mario Chiariello

Papers

Aiming for the brain: a new thermogel-based drug delivery platform

HrpA anchors meningococci to the dynein motor and affects the balance between apoptosis and pyroptosis

SNAI1 is upregulated during muscle regeneration and represses FGF21 and ATF3 expression by directly binding their promoters

HrpA anchors meningococci to the dynein motor and affects the balance between apoptosis and pyroptosis

MAPK15 controls mitochondrial fitness and contributes to prevent cellular senescence