Initially, the pandemic COVID-19, caused by SARS-CoV-2, was considered to be an exclusive lung disease, eventually leading to serious respiratory symptoms 1 . In the meantime, accumulating experimental and clinical studies have suggested that SARS-CoV-2 may also cause lesions in the kidneys, heart, brain, and gastrointestinal and endocrine organs [2][3][4][5][6][7] . SARS-CoV-2 tropism towards distinct tissues is governed by cellular factors expressed on target cells such as the viral entry receptor angiotensin-converting enzyme 2 (ACE2) 8 and the transmembrane serine protease 2 (TMPRSS2) 8 . ACE2 messenger RNA 9-13 and protein 12-14 expression within the islets of Langerhans has been reported, but not yet been shown, to allow SARS-CoV-2 entry 9,12,15 . Diabetes mellitus presents Janus like in 16 ): first, pre-existing diabetes is a highly prevalent comorbidity observed in 11-22% of patients and as such increases the risk of a severe disease, requiring more intense interventions and increasing mortality [17][18][19][20][21][22] . Second, SARS-CoV-2 infection seems to affect the exocrine pancreas, manifesting as pancreatitis in 32.5% of critically ill patients 23 , and pancreatic enlargement and abnormal amylase or lipase levels in 7.5-17% of patients 9,22 . Third, metabolic dysregulation has been observed in patients with COVID-19 as:(1) increased hyperglycaemia in patients with type 2 diabetes 24 ; (2) ketoacidosis in 2-6.4% of diabetic and non-diabetic patients 18,25 ; and (3), in case studies reporting ketoacidosis on SARS-CoV-2 infection, accompanied by (4) new-onset type 1 diabetes mellitus (T1DM) in the absence of autoantibodies [26][27][28] . In a cohort study of patients with diabetes, hyperglycaemia was reported in more than 50% of all cases, and almost a third experienced diabetic ketoacidosis 29 . Finally, a multicentre study found an 80% increase of new-onset T1DM in children during the COVID-19 pandemic 30 . In accordance, a recent meta-analysis summarizes that severe SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas

Dynamic actin remodelling processes at the leading edge of migrating tumour cells are concerted events controlled by a fine-tuned temporal and spatial interplay of kinases and phosphatases. Actin severing is regulated by ADF/Cofilin which regulates stimulus-induced lamellipodia protrusion and directed cell motility. Cofilin is activated by dephosphorylation via phosphatases of the slingshot (SSH) family. SSH activity is strongly increased by its binding to filamentous actin (F-actin), however, other upstream regulators remain unknown. We show that in response to RhoA activation, Protein Kinase D1 (PKD1) phosphorylates the SSH enzyme SSH1L at a serine residue located in its actin binding motif. This generates a 14-3-3 binding motif, blocks the localization of SSH1L to F-actin-rich structures in the lamellipodium by sequestering it in the cytoplasm. Consequently, expression of constitutively-active PKD1 in invasive tumour cells enhanced phosphorylation of cofilin and effectively blocked the formation of free actin filament barbed ends and directed cell migration.

Introduction The biological and molecular events that regulate the invasiveness of breast tumour cells need to be further revealed to develop effective therapies that stop breast cancer from expanding and metastasising.

ObjectiveThe generation of acinar and ductal cells from human pluripotent stem cells (PSCs) is a poorly studied process, although various diseases arise from this compartment.DesignWe designed a straightforward approach to direct human PSCs towards pancreatic organoids resembling acinar and ductal progeny.ResultsExtensive phenotyping of the organoids not only shows the appropriate marker profile but also ultrastructural, global gene expression and functional hallmarks of the human pancreas in the dish. Upon orthotopic transplantation into immunodeficient mice, these organoids form normal pancreatic ducts and acinar tissue resembling fetal human pancreas without evidence of tumour formation or transformation. Finally, we implemented this unique phenotyping tool as a model to study the pancreatic facets of cystic fibrosis (CF). For the first time, we provide evidence that in vitro, but also in our xenograft transplantation assay, pancreatic commitment occurs generally unhindered in CF. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) activation in mutated pancreatic organoids not only mirrors the CF phenotype in functional assays but also at a global expression level. We also conducted a scalable proof-of-concept screen in CF pancreatic organoids using a set of CFTR correctors and activators, and established an mRNA-mediated gene therapy approach in CF organoids.ConclusionsTaken together, our platform provides novel opportunities to model pancreatic disease and development, screen for disease-rescuing agents and to test therapeutic procedures.

We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein cortactin and the Arp actin polymerization machinery. PKD phosphorylates cortactin in vitro and in vivo at serine 298 thereby generating a 14-3-3 binding motif. In vitro, a phosphorylation-deficient cortactin-S298A protein accelerated VCA-Arp-cortactin-mediated synergistic actin polymerization and showed reduced F-actin binding, indicative of enhanced turnover of nucleation complexes. In vivo, cortactin co-localized with the nucleation promoting factor WAVE2, essential for lamellipodia extension, in the actin polymerization zone in Heregulin-treated MCF-7 cells. Using a 3-dye FRET-based approach we further demonstrate that WAVE2-Arp and cortactin prominently interact at these structures. Accordingly, cortactin-S298A significantly enhanced lamellipodia extension and directed cell migration. Our data thus unravel a previously unrecognized mechanism by which PKD controls cancer cell motility.The mechanistic elucidation of signaling pathways regulating dynamic actin remodeling processes in migrating cells is pivotal to a comprehensive understanding of cancer cell metastasis. Protein kinase D (PKD) 2 has recently been identified as a vital upstream regulator of polarized cell motility and F-actin organization (1-4). PKD localizes to sites of dynamic actin remodeling (1). The kinase activity is essential for the control of directed cell motility (1, 2), whereby active PKD1 inhibited, whereas kinase-inactive PKD1KD strongly enhanced motility and invasiveness (1-4). Mechanistically, a key role for PKD1 in controlling the activity of the ubiquitous F-actin depolymerizing-and severing factor cofilin via slingshot1L (SSH1L) cofilin phosphatase has been demonstrated. The activity of SSH1L is mainly regulated by its binding to filamentous actin (F-actin), which has been shown to strongly enhance its activity (5, 6). Phosphorylation of SSH1L at Ser 978 by active PKD1, e.g. downstream of RhoA or oxidative stress, generates a 14-3-3 binding motif within an important F-actin binding region, thus resulting in the sequestration of SSH1L away from dynamic actin structures, reducing SSH1 activity and active non-S3-phosphorylated cofilin levels (2). By severing actin filaments, cofilin increases both the availability of G-actin monomers as well as the number of "barbed ends" for polymerization (7). Furthermore, severed filaments are the preferred substrate for dendritic nucleation by the Arp complex (8, 9). Localized induction of actin polymerization and the formation of branched actin networks constitute the basis for membrane protrusion and cell motility (2, 10, 11). In line with an upstream regulatory role in the control of these processes, PKD1 and -2 are also capable of binding to F-actin in vitro (1). In the case of PKD1, in vivo F-actin binding has been demonstrated as well, which most likely facilitates an interaction with actin regulatory proteins such as SSH1L (2). We now have identified a second key regulatory signaling pathway ...

Protein kinase C (PKC), a family of lipid-activated serine kinases, is involved in multiple functions in the regulation of growth control. The PKC-related isoform PKC/PKD has been implicated in mitogenic signal cascades because of the activation of p42/p44 MAPK leading to Elk1-mediated gene transcription, and PKC/PKD has been shown to be activated via a PKC-dependent pathway. By using confocal analyses, we demonstrate here that PKC partially colocalizes with PKC in different cell types. Colocalization depends on the presence of the PKC pleckstrin homology domain. Coexpression of constitutively active PKC with PKC leads to a significant enhancement of the PKC substrate phosphorylation capacity as a result of an increased phosphorylation of the activation loop Ser 738/742 of PKC, whereas Ser 910 autophosphorylation remains unaffected. In vitro phosphorylation experiments show that PKC directly phosphorylates PKC on activation loop serines. Consequently, the p42 MAPK cascade is triggered leading to an increase in reporter gene activity driven by a serum-responsive element in HEK293 cells. At the same time, PKC-mediated JNK activation is reduced, providing evidence for a mutual regulation of PKC/PKC affecting different arms of the p38/ERK/ JNK pathways. Our data provide evidence for the sequential involvement of selective PKC isoforms in kinase cascades and identify the relevant domains in PKC for interaction with and activation by PKC as pleckstrin homology domain and activation loop.

SummaryCell migration and invasion are largely dependent on the complex organization of the various cytoskeletal components. Whereas the role of actin filaments and microtubules in cell motility is well established, the role of intermediate filaments in this process is incompletely understood. Organization and structure of the keratin cytoskeleton, which consists of heteropolymers of at least one type 1 and one type 2 intermediate filament, are in part regulated by post-translational modifications. In particular, phosphorylation events influence the properties of the keratin network. Sphingosylphosphorylcholine (SPC) is a bioactive lipid with the exceptional ability to change the organization of the keratin cytoskeleton, leading to reorganization of keratin filaments, increased elasticity, and subsequently increased migration of epithelial tumor cells. Here we investigate the signaling pathways that mediate SPC-induced keratin reorganization and the role of keratin phosphorylation in this process. We establish that the MEK-ERK signaling cascade regulates both SPC-induced keratin phosphorylation and reorganization in human pancreatic and gastric cancer cells and identify Ser431 in keratin 8 as the crucial residue whose phosphorylation is required and sufficient to induce keratin reorganization and consequently enhanced migration of human epithelial tumor cells.

The complement and neutrophil defense systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a, dose- and time-dependently resulted in a rapid C5a receptor-1 (C5aR1)-dependent shape-change, indicated by enhanced flow cytometric forward-scatter area values. Similar changes were observed after incubation with zymosan-activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high-resolution digital imaging revealed a C5a-induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluid optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a-induced shape changes were inhibited by pharmacological blockade of either the Cl−/HCO3−-exchanger or the Cl−-channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP–P2X/Y-purinoceptor interaction was also involved in the C5a-induced shape changes, because pre-treatment with suramin blocked not only the shape changes but also the subsequent C5a-dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin-cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.