Densely Populated Cell and Organelles Segmentation With Multi-Plane Deep Learning Pipeline.

Here we develop a robust machine vision pipeline for cell and organelle segmentation within vEM datasets. We collect neural network predictions along multiple planes, capturing 3D correlations using only 2D neural networks. We segment and analyze hundreds of platelets and report quantitative morphological measurements.

Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis.

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside.

Deep learning, 3D ultrastructural analysis reveals quantitative differences in platelet and organelle packing in COVID-19/SARSCoV2 patient-derived platelets.

Platelets contribute to COVID-19 clinical manifestations, of which microclotting in the pulmonary vasculature has been a prominent symptom. To investigate the potential diagnostic contributions of overall platelet morphology and their α-granules and mitochondria to the understanding of platelet hyperactivation and micro-clotting, we undertook a 3D ultrastructural approach. Because differences might be small, we used the high-contrast, high-resolution technique of focused ion beam scanning EM (FIB-SEM) and employed deep learning computational methods to evaluate nearly 600 individual platelets and 30 000 included organelles within three healthy controls and three severely ill COVID-19 patients.

A missense mouse model for Smith-McCort dysplasia shows bone resorption defects and altered protein glycosylation.

Smith McCort (SMC) dysplasia is a rare, autosomal recessive, osteochondrodysplasia that can be caused by pathogenic variants in either or genes. These genes codes for proteins that are located at the Golgi apparatus and have a role in intracellular vesicle trafficking. We generated mice that carry a disease-causing variant, c.

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis.

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like diabetes and obesity, and chronic inflammatory diseases like atherosclerosis, cancer, and autoimmune diseases. Upon vascular injury, usually the coagulation system, platelets, and endothelium act in an orchestrated manner to prevent bleeding by forming a clot at the site of the injury.

Tethered platelet capture provides a mechanism for restricting circulating platelet activation to the wound site.

Background: Puncture wounding is a longstanding challenge to human health for which understanding is limited, in part, by a lack of detailed morphological data on how the circulating platelet capture to the vessel matrix leads to sustained, self-limiting platelet accumulation.

Objectives: The objective of this study was to produce a paradigm for self-limiting thrombus growth in a mouse jugular vein model.

Methods: Data mining of advanced electron microscopy images was performed from authors' laboratories.

High-Pressure Freezing Followed by Freeze Substitution: An Optimal Electron Microscope Technique to Study Golgi Apparatus Organization and Membrane Trafficking.

A major goal of structural biologists is to preserve samples as close to their living state as possible. High-pressure freezing (HPF) is a state-of-art technique that freezes the samples at high pressure (~2100 bar) and low temperature (-196 °C) within milliseconds. This ultrarapid fixation enables simultaneous immobilization of all cellular components and preserves the samples in a near-native state.

Venous puncture wound hemostasis results in a vaulted thrombus structured by locally nucleated platelet aggregates.

Primary hemostasis results in a platelet-rich thrombus that has long been assumed to form a solid plug. Unexpectedly, our 3-dimensional (3D) electron microscopy of mouse jugular vein puncture wounds revealed that the resulting thrombi were structured about localized, nucleated platelet aggregates, pedestals and columns, that produced a vaulted thrombus capped by extravascular platelet adherence. Pedestal and column surfaces were lined by procoagulant platelets.

Dense cellular segmentation for EM using 2D-3D neural network ensembles.

Biologists who use electron microscopy (EM) images to build nanoscale 3D models of whole cells and their organelles have historically been limited to small numbers of cells and cellular features due to constraints in imaging and analysis. This has been a major factor limiting insight into the complex variability of cellular environments. Modern EM can produce gigavoxel image volumes containing large numbers of cells, but accurate manual segmentation of image features is slow and limits the creation of cell models.

Platelet α-granule cargo packaging and release are affected by the luminal proteoglycan, serglycin.

Background: Serglycin (SRGN) is an intragranular, sulfated proteoglycan in hematopoietic cells that affects granule composition and function.

Objective: To understand how SRGN affects platelet granule packaging, cargo release, and extra-platelet microenvironments.

Methods: Platelets and megakaryocytes from SRGN mice were assayed for secretion kinetics, cargo levels, granule morphology upon activation, and receptor shedding.

Rab6 is required for rapid, cisternal-specific, intra-Golgi cargo transport.

Rab6, the most abundant Golgi associated small GTPase, consists of 2 equally common isoforms, Rab6A and Rab6A', that differ in 3 amino acids and localize to trans Golgi cisternae. The two isoforms are largely redundant in function and hence are often referred to generically as Rab6. Rab6 loss-of-function inhibits retrograde Golgi trafficking, induces an increase in Golgi cisternal number in HeLa cells and delays the cell surface appearance of the anterograde cargo protein, VSVG.

Structural analysis of resting mouse platelets by 3D-EM reveals an unexpected variation in α-granule shape.

Mice and mouse platelets are major experimental models for hemostasis and thrombosis; however, important physiological data from this model has received little to no quantitative, 3D ultrastructural analysis. We used state-of-the-art, serial block imaging scanning electron microscopy (SBF-SEM, nominal Z-step size was 35 nm) to image resting platelets from C57BL/6 mice. α-Granules were identified morphologically and rendered in 3D space.

Canalicular system reorganization during mouse platelet activation as revealed by 3D ultrastructural analysis.

The canalicular system (CS) has been defined as: an inward, invaginated membrane connector that supports entry into and exit from the platelet; a static structure stable during platelet isolation; and the major source of plasma membrane (PM) for surface area expansion during activation. Recent analysis from STEM tomography and serial block face electron microscopy has challenged the relative importance of CS as the route for granule secretion. Here, We used 3D ultrastructural imaging to reexamine the CS in mouse platelets by generating high-resolution 3D reconstructions to test assumptions 2 and 3.

3D ultrastructural analysis of α-granule, dense granule, mitochondria, and canalicular system arrangement in resting human platelets.

Background: State-of-the-art 3-dimensional (3D) electron microscopy approaches provide a new standard for the visualization of human platelet ultrastructure. Application of these approaches to platelets rapidly fixed prior to purification to minimize activation should provide new insights into resting platelet ultrastructure.

Objectives: Our goal was to determine the 3D organization of α-granules, dense granules, mitochondria, and canalicular system in resting human platelets and map their spatial relationships.

Epistatic Analysis of the Contribution of Rabs and Kifs to CATCHR Family Dependent Golgi Organization.

Multisubunit members of the CATCHR family: COG and NRZ complexes, mediate intra-Golgi and Golgi to ER vesicle tethering, respectively. We systematically addressed the genetic and functional interrelationships between Rabs, Kifs, and the retrograde CATCHR family proteins: COG3 and ZW10, which are necessary to maintain the organization of the Golgi complex. We scored the ability of siRNAs targeting 19 Golgi-associated Rab proteins and all 44 human Kifs, microtubule-dependent motor proteins, to suppress CATCHR-dependent Golgi fragmentation in an epistatic fluorescent microscopy-based assay.

Autophagy in Platelets.

Anucleate platelets are produced by fragmentation of megakaryocytes. Platelets circulate in the bloodstream for a finite period: upon vessel injury, they are activated to participate in hemostasis; upon senescence, unused platelets are cleared. Platelet hypofunction leads to bleeding.

SNARE-dependent membrane fusion initiates α-granule matrix decondensation in mouse platelets.

Platelet α-granule cargo release is fundamental to both hemostasis and thrombosis. Granule matrix hydration is a key regulated step in this process, yet its mechanism is poorly understood. In endothelial cells, there is evidence for 2 modes of cargo release: a jack-in-the-box mechanism of hydration-dependent protein phase transitions and an actin-driven granule constriction/extrusion mechanism.

Alterations in platelet secretion differentially affect thrombosis and hemostasis.

We genetically manipulated the major platelet vesicle-associated membrane proteins (VAMP2, VAMP3, and VAMP8) to create mice with varying degrees of disrupted platelet secretion. As previously shown, loss of VAMP8 reduced granule secretion, and this defect was exacerbated by further deletion of VAMP2 and VAMP3. VAMP238 platelets also had reduced VAMP7.

GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells.

GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR) pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER)-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake.

Rab proteins as major determinants of the Golgi complex structure.

GTP-ases of the Rab family (about 70 in human) are key regulators of intracellular transport and membrane trafficking in eukaryotic cells. Remarkably, almost one third associate with membranes of the Golgi complex and TGN (trans-Golgi network). Through interactions with a variety of effectors that include molecular motors, tethering complexes, scaffolding proteins and lipid kinases, they play an important role in maintaining Golgi architecture.

Cellubrevin/vesicle-associated membrane protein-3-mediated endocytosis and trafficking regulate platelet functions.

Endocytosis is key to fibrinogen (Fg) uptake, trafficking of integrins (αIIbβ, αβ), and purinergic receptors (P2Y, P2Y), and thus normal platelet function. However, the molecular machinery required and possible trafficking routes are still ill-defined. To further identify elements of the platelet endocytic machinery, we examined the role of a vesicle-residing, soluble -ethylmaleimide factor attachment protein receptor (v-SNARE) called cellubrevin/vesicle-associated membrane protein-3 (VAMP-3) in platelet function.

Managing the Introduction of Super-Resolution Microscopy into a Core Facility.

Super resolution techniques place the resolution of fluorescence microscopy closer to the size of the underlying cell structure or molecular machine being studied. Structured illumination techniques will give users a set of tools that are close to their past experience and relatively simple and quick to learn. The present dyes can be used.

High-magnification super-resolution FINCH microscopy using birefringent crystal lens interferometers.

Fresnel incoherent correlation holography (FINCH) microscopy is a promising approach for high-resolution biological imaging but has so far been limited to use with low-magnification, low-numerical-aperture configurations. We report the use of in-line incoherent interferometers made from uniaxial birefringent α-barium borate (α-BBO) or calcite crystals that overcome the aberrations and distortions present with previous implementations that employed spatial light modulators or gradient refractive index lenses. FINCH microscopy incorporating these birefringent elements and high-numerical-aperture oil immersion objectives could outperform standard wide-field fluorescence microscopy, with, for example, a 149 nm lateral point spread function at a wavelength of 590 nm.

The cellular basis of platelet secretion: Emerging structure/function relationships.

Platelet activation has long been known to be accompanied by secretion from at least three types of compartments. These include dense granules, the major source of small molecules; α-granules, the major protein storage organelle; and lysosomes, the site of acid hydrolase storage. Despite ~60 years of research, there are still many unanswered questions about the cell biology of platelet secretion: for example, how are these secretory organelles organized to support cargo release and what are the key routes of cargo release, granule to plasma membrane or granule to canalicular system.