Protocols for Generating Surfaces and Measuring 3D Organelle Morphology Using Amira

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

Opto-Katanin: An Optogenetic Tool for Localized Microtubule Disassembly

Microtubules are major cytoskeletal filaments that drive chromosome separation during cell division, serve as rails for intracellular transport and as a scaffold for organelle positioning. Experimental manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubule integrity are currently lacking. Here, we exploit the dependence of the mammalian microtubule-severing protein katanin on microtubule-targeting co-factors to generate a light-activated system for localized microtubule disassembly that we named opto-katanin. Targeted illumination with blue light induces rapid and localized opto-katanin recruitment and local microtubule depolymerization, which is quickly reversible after stopping light-induced activation. Opto-katanin can be employed to locally perturb microtubule-based transport and organelle morphology in dividing cells and differentiated neurons with high spatiotemporal precision. We show that different microtubule-associated proteins can be used to recruit opto-katanin to microtubules and induce severing, paving the way for spatiotemporally precise manipulation of specific microtubule subpopulations.

Practices for Measuring 3D Organelle Morphology and Generating Surfaces with Amira

Analysis of 3D structures is of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have remained staples for imaging cellular structures, they lack the ability to image in 3D. However, recent technological advances, such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), have allowed researchers to observe cellular ultrastructure in 3D. Here, we propose a standardized protocol using the visualization software Amira to quantify organelle morphologies in 3D; this method allows researchers to produce accurate and reproducible measurements of cellular structure characteristics. We demonstrate this applicability by utilizing SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures.

P–063 The influence of lifestyle factors on sperm quality by motile sperm organelle morphology examination (MSOME)

Study question This study aimed to investigate the influence of lifestyle factors on sperm quality according to Motile Sperm Organelle Morphology Examination (MSOME) criteria. Summary answer The introduction of MSOME permits the examination of subcellular defect like nuclear vacuoles at hight magnification (6000x) in real time on vital sperm. What is known already It is increased accepted that lifestyle factors have an impact on sperm quality. Recent evidence shows that the selection of spermatozoa based on the analysis of morphology under high magnification may have a positive impact on embryo development in cases with severe male factor infertility and/or previous implantation failures. Therefore, MSOME has been considered as representing an improvement in the evaluation of semen quality. Although numerous studies have shown the influences of nutrition, lifestyle, age on semen quality, only very few study, have considered the influence of these factors on the vacuolization rate in semen analysis (MSOME criteria) Study design, size, duration The objective of this prospective study was to compare the semen parameters of 87 male patients undergoing evaluation or treatment of infertility at Unit of Pathophysiology of Reproduction ad PMA-Santa Maria Goretti Hospital -Latina according to MSOME and WHO (World Health Organization) criteria between January and September 2019.Written informed consent was obtained from all partecipant of this study. Participants/materials, setting, methods The subjects were divided into three groups according to age: Group 1 ≤ 35 yearş group II ,36–40 years; and Group III ≥ to 41 years .All patients filled a questionnaire answering questions regarding age, BMI, caffeine and alcohol consumptions, smoking and nutrition behavior. Were excluded from the study patients with chromosomal alteration. For multifactorial lifestyle influence patients were evaluated with a point base system with a cut-off >2 and cut off<3 for unhealthy style. Main results and the role of chance There was no difference between the groups with regard all semen parameters such as volume, concentration, number of leukocytes, morphology and vitality (%).The percentage of spermatozoa with LNV (Large Nuclear vacuoles) was significantly higher in the older group than in the younger (I and II) (39,14±13,74 vs 31,8±12 and 31,7 ±13,4 rispectively (p < 0,05)which does not correspond to a worsening of semen morphology. Regression analysis demonstrated a correlation between the percentage of spermatozoa with LNV and male age (r = –0,1) (p < 0,001). There was no correlation between lifestyle parameters ad enviroments factors . Comparing the semen parameters of healthy and unhealthy population we found no difference except a significantly higher number of spermatozoa with vacuoles in the unhealthy population (p < 0.001) Limitations, reasons for caution Although the sample examined in this study is limited in size and other studies are needed to confirm this evidence, the data available to us support the routine use of MSOME for ICSI and as a criterion for semen analysis with potential clinical repercussions. Wider implications of the findings: To date, there are few works in the literature that analyze the relationship between the morphology assessed with the MSOME and the age of the patients and the results are conflicting. To our knowledge many works agree with our results. Trial registration number Not applicable

Light at the end of the tunnel: FRAP assay reveals that plant vacuoles start as a tubular network

Plant vacuoles play key roles in cellular homeostasis performing catabolic and storage functions, regulating pH and ion balance 1,2. The essential role of vacuoles for plant cell viability makes them a notoriously difficult subject to study. As a consequence, there is still no consensus on the mechanism of vacuolar establishment and the source of membrane material for it. Our previous suggestion of endoplasmic reticulum (ER) being the main contributor of membrane for growing young vacuoles3 was recently challenged in a study proposing that plant vacuoles are formed de novo via homotypic fusion of multivesicular bodies (MVBs)4. Authors of this work pointed out issues that might explain our seemingly contradictory observations and we have thus carefully revaluated our hypothesis. Using the Arabidopsis thaliana root as a model, we provide a systematic overview of successive vacuolar biogenesis stages, starting from the youngest cells proximate to the quiescent center. We validate our previous conclusions by demonstrating that the vacuolar dye BCECF is fully suitable for studying the organelle morphology and provide 3D models from vacuoles of all developmental stages. We established a customized FRAP assay and proved that even at the earliest stages of biogenesis, vacuoles comprise a connected network. Together, this adds to a growing body of evidence indicating that vacuolar structures cannot originate solely from MVBs.

Biomembranes undergo complex, non-axisymmetric deformations governed by Kirchhoff-Love kinematics and revealed by a three dimensional computational framework

Biomembranes play a central role in various phenomena like locomotion of cells, cell-cell interactions, packaging and transport of nutrients, transmission of nerve impulses, and in maintaining organelle morphology and functionality. During these processes, the membranes undergo significant morphological changes through deformation, scission, and fusion. Modeling the underlying mechanics of such morphological changes has traditionally relied on reduced order axisymmetric representations of membrane geometry and deformation. Axisymmetric representations, while robust and extensively deployed, suffer from their inability to model symmetry breaking deformations and structural bifurcations. To address this limitation, a three-dimensional computational mechanics framework for high fidelity modeling of biomembrane deformation is presented. The proposed framework brings together Kirchhoff-Love thin-shell kinematics, Helfrich-energy based mechanics, and state-of-the-art numerical techniques for modeling deformation of surface geometries. Lipid bilayers are represented as spline-based surface discretizations immersed in a three-dimensional space; this enables modeling of a wide spectrum of membrane geometries, boundary conditions, and deformations that are physically admissible in a 3D space. The mathematical basis of the framework and its numerical machinery are presented, and their utility is demonstrated by modeling three classical, yet non-trivial, membrane deformation problems: formation of tubular shapes and their lateral constriction, Piezo1-induced membrane footprint generation and gating response, and the budding of membranes by protein coats during endocytosis. For each problem, the full three dimensional membrane deformation is captured, potential symmetry-breaking deformation paths identified, and various case studies of boundary and load conditions are presented. Using the endocytic vesicle budding as a case study, we also present a “phase diagram” for its symmetric and broken-symmetry states.

IDH1 mutations induce organelle defects via dysregulated phospholipids

Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.

Calcium Signaling and Mitochondrial Function in Presenilin 2 Knock-Out Mice: Looking for Any Loss-of-Function Phenotype Related to Alzheimer’s Disease

Alzheimer′s disease (AD) is the most common age-related neurodegenerative disorder in which learning, memory and cognitive functions decline progressively. Familial forms of AD (FAD) are caused by mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2) genes. Presenilin 1 (PS1) and its homologue, presenilin 2 (PS2), represent, alternatively, the catalytic core of the γ-secretase complex that, by cleaving APP, produces neurotoxic amyloid beta (Aβ) peptides responsible for one of the histopathological hallmarks in AD brains, the amyloid plaques. Recently, PSEN1 FAD mutations have been associated with a loss-of-function phenotype. To investigate whether this finding can also be extended to PSEN2 FAD mutations, we studied two processes known to be modulated by PS2 and altered by FAD mutations: Ca2+ signaling and mitochondrial function. By exploiting neurons derived from a PSEN2 knock-out (PS2–/–) mouse model, we found that, upon IP3-generating stimulation, cytosolic Ca2+ handling is not altered, compared to wild-type cells, while mitochondrial Ca2+ uptake is strongly compromised. Accordingly, PS2–/– neurons show a marked reduction in endoplasmic reticulum–mitochondria apposition and a slight alteration in mitochondrial respiration, whereas mitochondrial membrane potential, and organelle morphology and number appear unchanged. Thus, although some alterations in mitochondrial function appear to be shared between PS2–/– and FAD-PS2-expressing neurons, the mechanisms leading to these defects are quite distinct between the two models. Taken together, our data appear to be difficult to reconcile with the proposal that FAD-PS2 mutants are loss-of-function, whereas the concept that PS2 plays a key role in sustaining mitochondrial function is here confirmed.

Hypercaloric diets impair ovarian follicular development by inducing hyperinsulinemia and hyperlipidemia in female mice

Long-term hypercaloric diets adversely impact ovarian follicular development and fecundity. We investigated the effects of high sugar (HS), high fat low sugar (HFLS), and high fat normal sugar (HFNS) diets on ovarian follicular development by feeding mice for up to 180 days. Body weight, gonadal fat, glucose, lipid, insulin, estrous cycle, sex hormones, ovarian tissues, and follicle ultrastructure were examined, and the expression of metabolism-related proteins was evaluated immunoblotting in ovarians. The mice on hypercaloric diets showed hyperinsulinemia and hyperlipidemia and exhibited heavier body and gonadal fat weights, longer estrous cycles, and fewer numbers of preantral and antral follicles; and the follicles that did form had impaired organelle morphology. The sex hormone levels in blood were similar to controls, excepting significantly elevated estradiol levels for the HS diet. In ovarian tissues, AMPKα phosphorylation was reduced while AKT phosphorylation and caspase-3 were increased in ovarian tissues in mice on all three hypercaloric diets. The data from our study collectively indicates possible mechanisms through which long-term exposure to unhealthy hypercaloric diets may impair ovarian follicular development: hyperinsulinemia and hyperlipidemia.