Mechanisms and roles of podosomes and invadopodia.

Cell invasion into the surrounding extracellular matrix or across tissue boundaries and endothelial barriers occurs in both physiological and pathological scenarios such as immune surveillance or cancer metastasis. Podosomes and invadopodia, collectively called 'invadosomes', are actin-based structures that drive the proteolytic invasion of cells, by forming highly regulated platforms for the localized release of lytic enzymes that degrade the matrix. Recent advances in high-resolution microscopy techniques, in vivo imaging and high-throughput analyses have led to considerable progress in understanding mechanisms of invadosomes, revealing the intricate inner architecture of these structures, as well as their growing repertoire of functions that extends well beyond matrix degradation. In this Review, we discuss the known functions, architecture and regulatory mechanisms of podosomes and invadopodia. In particular, we describe the molecular mechanisms of localized actin turnover and microtubule-based cargo delivery, with a special focus on matrix-lytic enzymes that enable proteolytic invasion. Finally, we point out topics that should become important in the invadosome field in the future.

Matrix metalloproteinases at a glance.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that belong to the group of endopeptidases or matrixins. They are able to cleave a plethora of substrates, including components of the extracellular matrix and cell-surface-associated proteins, as well as intracellular targets. Accordingly, MMPs play key roles in a variety of physiological and pathological processes, such as tissue homeostasis and cancer cell invasion. MMP activity is exquisitely regulated at several levels, including pro-domain removal, association with inhibitors, intracellular trafficking and transport via extracellular vesicles. Moreover, the regulation of MMP activity is currently being rediscovered for the development of respective therapies for the treatment of cancer, as well as infectious, inflammatory and neurological diseases. In this Cell Science at a Glance article and the accompanying poster, we present an overview of the current knowledge regarding the regulation of MMP activity, the intra- and extra-cellular trafficking pathways of these enzymes and their diverse groups of target proteins, as well as their impact on health and disease.

Degrading devices: invadosomes in proteolytic cell invasion.

Podosomes and invadopodia, collectively known as invadosomes, are cell-matrix contacts in a variety of cell types, such as monocytic cells or cancer cells, that have to cross tissue barriers. Both structures share an actin-rich core, which distinguishes them from other matrix contacts, and are regulated by a multitude of signaling pathways including RhoGTPases, kinases, actin-associated proteins, and microtubule-dependent transport. Invadosomes recruit and secrete proteinases and are thus able to lyse extracellular matrix components. They are therefore considered to be potential key structures in proteolytic cell invasion in both physiological and pathological settings. This review provides an overview of the field, with special focus on current developments such as intracellular transport processes, ultrastructural analysis, the possible involvement of invadosomes in disease, and the tentative identification of invadosomes in 3D environments and in vivo.

Endoscopic main duct stenting in refractory postoperative pancreatic fistula after distal pancreatectomy - a friend or a foe?

BACKGROUND: Clinically relevant (CR) postoperative pancreatic fistula (POPF) after distal pancreatectomy (DP) are common. Endoscopic treatment (ET) has only scarcely been explored. The aim of this study was to evaluate risk factors for CR POPF after DP and the efficacy of ET in adjunct to standard therapy. METHODS: Consecutive patients without previous pancreatic surgery who underwent DP between 2011 and 2020 were evaluated, analyzing risk factors for CR POPF. The choice and performance of ET, main pancreatic duct (MPD) stenting, was not standardized. Healing time and complications after ET were registered. RESULTS: 406 patients underwent DP, CR POPF occurred in 29.6%. ET was performed in 17 patients 27 days (median) after index surgery. Risk for CR POPF was increased in ASA-PS 1-2 patients, MPD ≤ 3 mm, procedure time ≥ 3 h, and CRP ≥ 180 on postoperative day 3. POPF resolved with standard treatment after 32 days and 59 days in the ET group (p < 0.001). There was one mortality in the ET-group (not procedure related). Mild post-ERCP pancreatitis occurred in three patients. CONCLUSIONS: CR POPF is common after DP. Long operating time, a narrow MPD, low ASA score, and high postoperative CRP were risk factors for CR POPF. ET was not beneficial but proper evaluation was not possible due to few patients and non-standardized treatment. Complications after ET appeared mild.

FIB-SEM-based analysis of Borrelia intracellular processing by human macrophages.

Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, joints and nervous system. Successful internalization and intracellular processing of borreliae by immune cells, like macrophages, is decisive for the outcome of a respective infection. Here, we use, for the first time, focused ion beam scanning electron microscopy tomography (FIB-SEM tomography) to visualize the interaction of borreliae with primary human macrophages with high resolution. We report that interaction between macrophages and the elongated and highly motile borreliae can lead to formation of membrane tunnels that extend deeper into the host cytoplasm than the actual phagosome, most probably as a result of partial extrication of captured borreliae. We also show that membrane tubulation at borreliae-containing phagosomes, a process suggested earlier as a mechanism leading to phagosome compaction but hard to visualize in live-cell imaging, is apparently a frequent phenomenon. Finally, we demonstrate that the endoplasmic reticulum (ER) forms multiple STIM1-positive contact sites with both membrane tunnels and phagosome tubulations, confirming the important role of the ER during uptake and intracellular processing of borreliae.

Poji: a Fiji-based tool for analysis of podosomes and associated proteins.

Podosomes are actin-based adhesion and invasion structures in a variety of cell types, with podosome-forming cells displaying up to several hundreds of these structures. Podosome number, distribution and composition can be affected by experimental treatments or during regular turnover, necessitating a tool that is able to detect even subtle differences in podosomal properties. Here, we present a Fiji-based macro code termed 'Poji' ('podosome analysis by Fiji'), which serves as an easy-to-use tool to characterize a variety of cellular and podosomal parameters, including area, fluorescence intensity, relative enrichment of associated proteins and radial podosome intensity profiles. This tool should be useful to gain more detailed insight into the regulation, architecture and functions of podosomes. Moreover, we show that Poji is easily adaptable for the analysis of invadopodia and associated extracellular matrix degradation, and likely also of other micron-size punctate structures. This article describes the workflow of the Poji macro, presents several examples of its applications, and also points out limitations, as well as respective solutions, and adaptable features to streamline the analysis.This article has an associated First Person interview with the first author of the paper.

Impact of spatio-temporal recurrence pattern on overall survival for invasive intraductal papillary mucinous neoplasia - A comparison with pancreatic ductal adenocarcinoma.

BACKGROUND: Resections for intraductal papillary mucinous neoplasia (IPMN) have increased dramatically during the last decade. Recurrence pattern and impact of adjuvant chemotherapy for solid pancreatic ductal adenocarcinoma (PDAC) is well known, but not for invasive IPMN (inv-IPMN). OBJECTIVES: To elucidate the impact of spatio-temporal recurrence pattern and adjuvant chemotherapy on overall survival for inv-IPMN compared with PDAC. METHODS: We conducted a retrospective single-center observational study of consecutive patients ≥18 years of age who underwent resection for inv-IPMN or PDAC at Karolinska University Hospital, between 2009 and 2018. Different initial recurrence sites and time frames as well as predictors for death were assessed with multivariable Cox and logistic regressions. Survival analyses were performed using the Kaplan-Meier model and log rank test. RESULTS: Of 396 resected patients, 92 were inv-IPMN and 304 PDAC. Both recurrence rate and death rate within three-years were lower for inv-IPMN compared to PDAC (p = 0.006 and p = 0.007 respectively). Across the whole cohort, the most common recurrence patterns were multi-site (25%), single-site liver (21%) and single-site locoregional (10%) recurrence. The most prominent predictors for death in multivariable Cox regression, especially if occurred within the first year, were multi-site (HR 17.0), single-site peritoneal (HR 13.6) and single-site liver (HR 13.1) recurrence. These predictors were less common in inv-IPMN compared to PDAC (p = 0.007). The effect of adjuvant chemotherapy was similar in the two groups. CONCLUSION: Resected inv-IPMN exhibits a less aggressive recurrence pattern than PDAC that translates into a more favorable overall survival.

Probing the mechanical landscape - new insights into podosome architecture and mechanics.

Podosomes are dynamic adhesion structures formed constitutively by macrophages, dendritic cells and osteoclasts and transiently in a wide variety of cells, such as endothelial cells and megakaryocytes. They mediate numerous functions, including cell-matrix adhesion, extracellular matrix degradation, mechanosensing and cell migration. Podosomes present as micron-sized F-actin cores surrounded by an adhesive ring of integrins and integrin-actin linkers, such as talin and vinculin. In this Review, we highlight recent research that has considerably advanced our understanding of the complex architecture-function relationship of podosomes by demonstrating that the podosome ring actually consists of discontinuous nano-clusters and that the actin network in between podosomes comprises two subsets of unbranched actin filaments, lateral and dorsal podosome-connecting filaments. These lateral and dorsal podosome-connecting filaments connect the core and ring of individual podosomes and adjacent podosomes, respectively. We also highlight recent insights into the podosome cap as a novel regulatory module of actomyosin-based contractility. We propose that these newly identified features are instrumental for the ability of podosomes to generate protrusion forces and to mechanically probe their environment. Furthermore, these new results point to an increasing complexity of podosome architecture and have led to our current view of podosomes as autonomous force generators that drive cell migration.

MT1-MMP targeting to endolysosomes is mediated by upregulation of flotillins.

Tumor cell invasion and metastasis formation are the major cause of death in cancer patients. These processes rely on extracellular matrix (ECM) degradation mediated by organelles termed invadopodia, to which the transmembrane matrix metalloproteinase MT1-MMP (also known as MMP14) is delivered from its reservoir, the RAB7-containing endolysosomes. How MT1-MMP is targeted to endolysosomes remains to be elucidated. Flotillin-1 and -2 are upregulated in many invasive cancers. Here, we show that flotillin upregulation triggers a general mechanism, common to carcinoma and sarcoma, which promotes RAB5-dependent MT1-MMP endocytosis and its delivery to RAB7-positive endolysosomal reservoirs. Conversely, flotillin knockdown in invasive cancer cells greatly reduces MT1-MMP accumulation in endolysosomes, its subsequent exocytosis at invadopodia, ECM degradation and cell invasion. Our results demonstrate that flotillin upregulation is necessary and sufficient to promote epithelial and mesenchymal cancer cell invasion and ECM degradation by controlling MT1-MMP endocytosis and delivery to the endolysosomal recycling compartment.

Structural and Functional Analyses of the Shedding Protease ADAM17 in HoxB8-Immortalized Macrophages and Dendritic-like Cells.

A disintegrin and metalloproteinase (ADAM) 17 has been implicated in many shedding processes. Major substrates of ADAM17 are TNF-α, IL-6R, and ligands of the epidermal growth factor receptor. The essential role of the protease is emphasized by the fact that ADAM17 deficiency is lethal in mice. To study ADAM17 function in vivo, we generated viable hypomorphic ADAM17 mice called ADAM17ex/ex mice. Recent studies indicated regulation of proteolytic ADAM17 activity by cellular processes such as cytoplasmic phosphorylation and removal of the prodomain by furin cleavage. Maturation and thus activation of ADAM17 is not fully understood. So far, studies of ADAM17 maturation have been mainly limited to mouse embryonic fibroblasts or transfected cell lines relying on nonphysiologic stimuli such as phorbol esters, thus making interpretation of the results difficult in a physiologic context. In this article, we present a robust cell system to study ADAM17 maturation and function in primary cells of the immune system. To this end, HoxB8 conditionally immortalized macrophage precursor cell lines were derived from bone marrow of wild-type and hypomorphic ADAM17ex/ex mice, which are devoid of measurable ADAM17 activity. ADAM17 mutants were stably expressed in macrophage precursor cells, differentiated to macrophages under different growth factor conditions (M-CSF versus GM-CSF), and analyzed for cellular localization, proteolytic activity, and podosome disassembly. Our study reveals maturation and activity of ADAM17 in a more physiological-immune cell system. We show that this cell system can be further exploited for genetic modifications of ADAM17 and for studying its function in immune cells.

ERCP-related perforations: a population-based study of incidence, mortality, and risk factors.

BACKGROUND: Perforations related to endoscopic retrograde cholangiopancreatography (ERCP) are rare but feared adverse events with highly reported morbidity and mortality rates. The aim was to evaluate the incidence and outcome of ERCP-related perforations and to identify risk factors for death due to perforations in a population-based study. METHODS: Between May 2005 and December 2013, a total of 52,140 ERCPs were registered in GallRiks, a Swedish nationwide, population-based registry. A total of 376 (0.72%) were registered as perforations or extravasation of contrast during ERCP or as perforation in the 30-day follow-up. The patients with perforation were divided into fatal and non-fatal groups and analyzed for mortality risk factors. The case volume of centers and endoscopists were divided into the upper quartile (Q4) and the lower three quartile (Q1-3) groups. Furthermore, fatal group patients' records were reviewed. RESULTS: Death within 90 days after ERCP-related perforations or at the index hospitalization occurred in 20% (75 out of 376) for all perforations and 0.1% (75 out of 52,140) for all ERCPs. The independent risk factors for death after perforation were malignancy (OR 11.2, 95% CI 5.8-21.6), age over 80 years (OR 3.8, 95% CI 2.0-7.4), and sphincterotomy in the pancreatic duct (OR 2.8, 95% CI 1.1-7.5). In Q4 centers, the mortality was similar with or without pancreatic duct sphincterotomy (14% vs. 13%, p = 1.0), but in Q1-3 centers mortality was higher (45% vs. 21%, p = 0.024). CONCLUSIONS: ERCP-related perforations are severe adverse events with low incidence (0.7%) and high mortality rate up to 20%. Malignancy, age over 80 years, and sphincterotomy in the pancreatic duct increase the risk to die after a perforation. The risk of a fatal outcome in perforations after pancreatic duct sphincterotomy was reduced when occurred at a Q4-center. In the case of a complicated perforation a transfer to a Q4-center may be considered.

Actin assembly mechanisms at a glance.

The actin cytoskeleton and associated motor proteins provide the driving forces for establishing the astonishing morphological diversity and dynamics of mammalian cells. Aside from functions in protruding and contracting cell membranes for motility, differentiation or cell division, the actin cytoskeleton provides forces to shape and move intracellular membranes of organelles and vesicles. To establish the many different actin assembly functions required in time and space, actin nucleators are targeted to specific subcellular compartments, thereby restricting the generation of specific actin filament structures to those sites. Recent research has revealed that targeting and activation of actin filament nucleators, elongators and myosin motors are tightly coordinated by conserved protein complexes to orchestrate force generation. In this Cell Science at a Glance article and the accompanying poster, we summarize and discuss the current knowledge on the corresponding protein complexes and their modes of action in actin nucleation, elongation and force generation.

Differences in Shedding of the Interleukin-11 Receptor by the Proteases ADAM9, ADAM10, ADAM17, Meprin α, Meprin ß and MT1-MMP.

Interleukin-11 (IL-11) has been associated with inflammatory conditions, bone homeostasis, hematopoiesis, and fertility. So far, these functions have been linked to classical IL-11 signaling via the membrane bound receptor (IL-11R). However, a signaling cascade via the soluble IL-11R (sIL-11R), generated by proteolytic cleavage, can also be induced. This process is called IL-11 trans-signaling. A disintegrin and metalloprotease 10 (ADAM10) and neutrophil elastase were described as ectodomain sheddases of the IL-11R, thereby inducing trans-signaling. Furthermore, previous studies employing approaches for the stimulation and inhibition of endogenous ADAM-proteases indicated that ADAM10, but not ADAM17, can cleave the IL-11R. Herein, we show that several metalloproteases, namely ADAM9, ADAM10, ADAM17, meprin ß, and membrane-type 1 matrix metalloprotease/matrix metalloprotease-14 (MT1-MMP/MMP-14) when overexpressed are able to shed the IL-11R. All sIL-11R ectodomains were biologically active and capable of inducing signal transducer and activator of transcription 3 (STAT3) phosphorylation in target cells. The difference observed for ADAM10/17 specificity compared to previous studies can be explained by the different approaches used, such as stimulation of protease activity or making use of cells with genetically deleted enzymes.

The formins FHOD1 and INF2 regulate inter- and intra-structural contractility of podosomes.

Podosomes are actin-rich adhesion structures that depend on Arp2/3-complex-based actin nucleation. We now report the identification of the formins FHOD1 and INF2 as novel components and additional actin-based regulators of podosomes in primary human macrophages. FHOD1 surrounds the podosome core and is also present at podosome-connecting cables, whereas INF2 localizes at the podosome cap structure. Using a variety of microscopy-based methods; including a semiautomated podosome reformation assay, measurement of podosome oscillations, FRAP analysis of single podosomes, and structured illumination microscopy, both formins were found to regulate different aspects of podosome-associated contractility, with FHOD1 mediating actomyosin contractility between podosomes, and INF2 regulating contractile events at individual podosomes. Moreover, INF2 was found to be a crucial regulator of podosome de novo formation and size. Collectively, we identify FHOD1 and INF2 as novel regulators of inter- and intra-structural contractility of podosomes. Podosomes thus present as one of the few currently identified structures which depend on the concerted activity of both Arp2/3 complex and specific formins and might serve as a model system for the analysis of complex actin architectures in cells.

Actin-Dependent Regulation of Borrelia burgdorferi Phagocytosis by Macrophages.

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, nervous system, and joints. If an infection with Borrelia proceeds unchecked, the disease can also enter a chronic stage, leading to the development of neuroborreliosis or cardiac arrhythmia. Successful elimination of B. burgdorferi by the host immune system is thus decisive for the positive outcome of a respective infection. Accordingly, host immune cells such as macrophages and dendritic cells have to be able to efficiently internalize and degrade infecting spirochetes. These processes are based on closely controlled rearrangements of the actin cytoskeleton, which enables the spatiotemporally fine-tuned formation of cellular protrusions and compartments that assist in the capturing, immobilization, and uptake of borreliae, as well as their further intracellular processing. Here, we discuss actin-based structures, in particular filopodia and coiling pseudopods that are involved in phagocytosis of B. burgdorferi by macrophages, their regulation by actin-associated proteins such as formins and Arp2/3 complex, as well as the subsequent intracellular processing of borreliae.

The Essential Elements of a Risk Governance Framework for Current and Future Nanotechnologies.

Societies worldwide are investing considerable resources into the safe development and use of nanomaterials. Although each of these protective efforts is crucial for governing the risks of nanomaterials, they are insufficient in isolation. What is missing is a more integrative governance approach that goes beyond legislation. Development of this approach must be evidence based and involve key stakeholders to ensure acceptance by end users. The challenge is to develop a framework that coordinates the variety of actors involved in nanotechnology and civil society to facilitate consideration of the complex issues that occur in this rapidly evolving research and development area. Here, we propose three sets of essential elements required to generate an effective risk governance framework for nanomaterials. (1) Advanced tools to facilitate risk-based decision making, including an assessment of the needs of users regarding risk assessment, mitigation, and transfer. (2) An integrated model of predicted human behavior and decision making concerning nanomaterial risks. (3) Legal and other (nano-specific and general) regulatory requirements to ensure compliance and to stimulate proactive approaches to safety. The implementation of such an approach should facilitate and motivate good practice for the various stakeholders to allow the safe and sustainable future development of nanotechnology.

Feel the force: Podosomes in mechanosensing.

Cells interact with their environment through highly localized contact structures. Podosomes represent a subgroup of cell-matrix contacts, which is especially prominent in cells of the monocytic lineage such as monocytes, macrophages and dendritic cells, but also in a variety of other cell types. Comparable to other adhesion structures, podosomes feature a complex architecture, which forms the basis for their extensive repertoire of sensory and effector functions. These functions are mainly linked to interactions with the extracellular matrix and comprise well known properties such as cell-matrix adhesion and extracellular matrix degradation. A more recent discovery is the ability of podosomes to act as mechanosensory devices, by detecting rigidity and topography of the substratum. In this review, we focus especially on the molecular events involved in mechanosensing by podosomes, the structural elements of podosomes that enable this function, as well as the intra- and extracellular signals generated downstream of podosome mechanosensing.

Drebrin's Role in the Maintenance of Endothelial Integrity.

The human endothelium forms a permeable barrier between the blood stream and surrounding tissues, strictly governing the passage of immune cells, fluids and metabolites. The regulation of cell-cell contact dynamics between endothelial cells is essential for this function and thus for the maintenance of vascular integrity. Intercellular adhesion within the endothelium is mainly dependent on adherens junctions, composed of cell-cell adhesion proteins such as VE-cadherin and nectin, and their associated proteins. Recent research points to a critical role of the actin cytoskeleton in endothelial integrity, by providing anchorage of adhesion complexes to the cell cortex. We could show that the F-actin-binding protein drebrin is a critical regulator of endothelial integrity, by linking nectin to the cortical actin cytoskeleton. In particular, the knockdown of drebrin leads to functional impairment of endothelial cells, characterized by rupturing of endothelial monolayers cultured under conditions mimicking vascular flow. This weakening of cell-cell contacts upon drebrin depletion is based on the destabilization of nectin at adherens junctions, followed by internalization and degradation in lysosomes. Conducting interaction studies, we showed that drebrin binds to nectin's interaction partner afadin, thus linking the nectin/afadin system to the cortical F-actin network. Drebrin, containing binding sites for both afadin and F-actin, is thus uniquely equipped to stabilize nectin at adherens junctions, thereby preserving endothelial integrity. Collectively, these results contribute to the current understanding of cell-cell junction regulation, introducing a new function of drebrin as a stabilizer of endothelial integrity.

Tools of the trade: podosomes as multipurpose organelles of monocytic cells.

Podosomes are adhesion and invasion structures that are particularly prominent in cells of the monocytic lineage such as macrophages, dendritic cells, and osteoclasts. They are multifunctional organelles that combine several key abilities required for cell migration and invasion. The podosome repertoire includes well-established functions such as cell-substrate adhesion, and extracellular matrix degradation, recently discovered abilities such as rigidity and topology sensing as well as antigen sampling, and also more speculative functions such as cell protrusion stabilization and transmigration. Collectively, podosomes not only enable dynamic interactions of cells with their surroundings, they also gather information about the pericellular environment, and are actively involved in its reshaping. This review presents an overview of the current knowledge on podosome composition, architecture, and regulation. We focus in particular on the growing list of podosome functions and discuss the specific properties of podosomes in macrophages, dendritic cells, and osteoclasts. Moreover, this article highlights podosome-related intracellular transport processes, the formation of podosomes in 3D environments as well as potentially podosome-associated diseases involving monocytic cells.

A specific subset of RabGTPases controls cell surface exposure of MT1-MMP, extracellular matrix degradation and three-dimensional invasion of macrophages.

The matrix metalloproteinase MT1-MMP has a major impact on invasive cell migration in both physiological and pathological settings such as immune cell extravasation or metastasis of cancer cells. Surface-associated MT1-MMP is able to cleave components of the extracellular matrix, which is a prerequisite for proteolytic invasive migration. However, current knowledge on the molecular mechanisms that regulate MT1-MMP trafficking to and from the cell surface is limited. We have identified three members of the RabGTPase family, Rab5a, Rab8a and Rab14, as crucial regulators of MT1-MMP trafficking and function in primary human macrophages. Both overexpressed and endogenous forms show prominent colocalisation with MT1-MMP-positive vesicles, whereas expression of mutant constructs, as well as siRNA-induced knockdown, reveal that these RabGTPases are crucial in the regulation of MT1-MMP surface exposure, contact of MT1-MMP-positive vesicles with podosomes, extracellular matrix degradation in two and three dimensions, as well as three-dimensional proteolytic invasion of macrophages. Collectively, our results identify Rab5a, Rab8a and Rab14 as major regulators of MT1-MMP trafficking and invasive migration of primary human macrophages, which could be promising potential targets for manipulation of immune cell invasion.