Search

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.

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 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 influence of external triggers (mechanical stimulation, drug administration).

Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Directly linking molecular to nanoscale ultrastructural information is therefore crucial to understand cellular physiology. Volume or 3-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. Application of volume-CLEM is however hampered by limitations in throughput and 3D correlation efficiency. Addressing these limitations, we here describe a novel pipeline for volume-CLEM that provides high-precision (SignificanceWe have developed a correlative imaging pipeline to (i) correlate 3D-FM to volume-EM data with high precision, directly bridging the FM and EM resolutions (ii) achieve high-throughput volume-CLEM by targeted EM imaging of a single organelle sized region-of-interest, pre-identified by FM (iii) link live-cell fluorescence imaging of cultured mammalian cells to high-throughput volume-CLEM (iv) quantitatively study structure-function relations at subcellular scale (v) link rare (e.g. membrane contact sites) and transient (e.g. organelle interactions) cellular events to 3D ultrastructure.The targeted volume-CLEM pipeline provides a unique prospect for multi-modal correlative intracellular analysis combining dynamic interaction (live-cell imaging), functional state (live-cell imaging), molecular localization (FM), and 3D-ultrastructure (FIB.SEM) at nanometer scale.