Loss of the HOPS complex disrupts early-to-late endosome transition, impairs endosomal recycling and induces accumulation of amphisomes

The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These 'HOPS bodies' were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation

Correlative Organelle Microscopy: Fluorescence Guided Volume Electron Microscopy of Intracellular Processes

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Therefore, directly linking molecular to nanoscale ultrastructural information is crucial in understanding cellular physiology. Volume or three-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. However, the application of volume-CLEM is hampered by limitations in throughput and 3D correlation efficiency. In order to address these limitations, we describe a novel pipeline for volume-CLEM that provides high-precision (<100 nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) datasets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated into a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume EM and obviates the need for post-correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between the endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D

An Organoid for Woven Bone

Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel, both cellular and matrix formation processes. Stem cell‐based organoids are promising as functional, self‐organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self‐organizing co‐culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in in vivo osteocytes, the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under the influence of external triggers (mechanical stimulation, drug administration)

EB1 and EB3 regulate microtubule minus end organization and Golgi morphology

End-binding proteins (EBs) are the core components of microtubule plus end tracking protein complexes, but it is currently unknown whether they are essential for mammalian microtubule organization. Here, by using CRISPR/Cas9-mediated knockout technology, we generated stable cell lines lacking EB2 and EB3 and the C-terminal partner-binding half of EB1. These cell lines show only mild defects in cell division and microtubule polymerization. However, the length of CAMSAP2-decorated stretches at noncentrosomal microtubule minus ends in these cells is reduced, microtubules are detached from Golgi membranes, and the Golgi complex is more compact. Coorganization of microtubules and Golgi membranes depends on the EB1/EB3-myomegalin complex, which acts as membrane-microtubule tether and counteracts tight clustering of individual Golgi stacks. Disruption of EB1 and EB3 also perturbs cell migration, polarity, and the distribution of focal adhesions. EB1 and EB3 thus affect multiple interphase processes and have a major impact on microtubule minus end organization

Quantitative 3D microscopy highlights altered von Willebrand factor α-granule storage in patients with von Willebrand disease with distinct pathogenic mechanisms

Background: Platelets play a key role in hemostasis through plug formation and secretion of their granule contents at sites of endothelial injury. Defects in von Willebrand factor (VWF), a platelet α-granule protein, are implicated in von Willebrand disease (VWD), and may lead to defective platelet adhesion and/or aggregation. Studying VWF quantity and subcellular localization may help us better understand the pathophysiology of VWD. Objective: Quantitative analysis of the platelet α-granule compartment and VWF storage in healthy individuals and VWD patients. Patients/Methods: Structured illumination microscopy (SIM) was used to study VWF content and organization in platelets of healthy individuals and patients with VWD in combination with established techniques. Results: SIM capably quantified clear morphological and granular changes in platelets stimulated with proteinase-activated receptor 1 (PAR-1) activating peptide and revealed a large intra- and interdonor variability in VWF-positive object numbers within healthy resting platelets, similar to variation in secreted protein acidic and rich in cysteine (SPARC). We subsequently characterized VWD platelets to identify changes in the α-granule compartment of patients with different VWF defects, and were able to stratify two patients with type 3 VWD rising from different pathological mechanisms. We further analyzed VWF storage in α-granules of a patient with homozygous p.C1190R using electron microscopy and found discrepant VWF levels and different degrees of multimerization in platelets of patients with heterozygous p.C1190 in comparison to VWF in plasma. Conclusions: Our findings highlight the utility of quantitative imaging approaches in assessing platelet granule content, which may help to better understand VWF storage in α-granules and to gain new insights in the etiology of VWD

Quantitative 3D microscopy highlights altered von Willebrand factor α-granule storage in patients with von Willebrand disease with distinct pathogenic mechanisms

Background: Platelets play a key role in hemostasis through plug formation and secretion of their granule contents at sites of endothelial injury. Defects in von Willebrand factor (VWF), a platelet α-granule protein, are implicated in von Willebrand disease (VWD), and may lead to defective platelet adhesion and/or aggregation. Studying VWF quantity and subcellular localization may help us better understand the pathophysiology of VWD. Objective: Quantitative analysis of the platelet α-granule compartment and VWF storage in healthy individuals and VWD patients. Patients/Methods: Structured illumination microscopy (SIM) was used to study VWF content and organization in platelets of healthy individuals and patients with VWD in combination with established techniques. Results: SIM capably quantified clear morphological and granular changes in platelets stimulated with proteinase-activated receptor 1 (PAR-1) activating peptide and revealed a large intra- and interdonor variability in VWF-positive object numbers within healthy resting platelets, similar to variation in secreted protein acidic and rich in cysteine (SPARC). We subsequently characterized VWD platelets to identify changes in the α-granule compartment of patients with different VWF defects, and were able to stratify two patients with type 3 VWD rising from different pathological mechanisms. We further analyzed VWF storage in α-granules of a patient with homozygous p.C1190R using electron microscopy and found discrepant VWF levels and different degrees of multimerization in platelets of patients with heterozygous p.C1190 in comparison to VWF in plasma. Conclusions: Our findings highlight the utility of quantitative imaging approaches in assessing platelet granule content, which may help to better understand VWF storage in α-granules and to gain new insights in the etiology of VWD.</p

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase

EGFR Dynamics Change during Activation in Native Membranes as Revealed by NMR

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase