Impaired mitochondrial morphological plasticity and failure of mitophagy associated with the G11778A mutation of LHON.

Mitochondrial dynamics were examined in human dermal fibroblasts biopsied from a confirmed Leber's Hereditary Optic Neuropathy (LHON) patient with a homoplasmic G11778A mutation of the mitochondrial genome. Expression of the G11778A mutation did not impart any discernible difference in mitochondrial network morphology using widefield fluorescence microscopy. However, at the ultrastructural level, cells expressing this mutation exhibited an impairment of mitochondrial morphological plasticity when forced to utilize oxidative phosphorylation (OXPHOS) by transition to glucose-free, galactose-containing media. LHON fibroblasts also displayed a transient increase in mitophagy upon transition to galactose media. These results provide new insights into the consequences of the G11778A mutation of LHON and the pathological mechanisms underlying this disease.

Podocalyxin is required for maintaining blood-brain barrier function during acute inflammation.

Podocalyxin (Podxl) is broadly expressed on the luminal face of most blood vessels in adult vertebrates, yet its function on these cells is poorly defined. In the present study, we identified specific functions for Podxl in maintaining endothelial barrier function. Using electrical cell substrate impedance sensing and live imaging, we found that, in the absence of Podxl, human umbilical vein endothelial cells fail to form an efficient barrier when plated on several extracellular matrix substrates. In addition, these monolayers lack adherens junctions and focal adhesions and display a disorganized cortical actin cytoskeleton. Thus, Podxl has a key role in promoting the appropriate endothelial morphogenesis required to form functional barriers. This conclusion is further supported by analyses of mutant mice in which we conditionally deleted a floxed allele of Podxl in vascular endothelial cells (vECs) using Tie2Cre mice (PodxlΔTie2Cre). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood-brain barrier (BBB) in PodxlΔTie2Cre mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH2) of the protease-activated receptor-1 (PAR-1), a thrombin receptor expressed by vECs, neuronal cells, and glial cells. In response to systemic administration of TFLLR-NH2, LPS-primed PodxlΔTie2Cre mice become completely immobilized for a 5-min period, coinciding with severely dampened neuroelectric activity. We conclude that Podxl expression by CNS tissue vECs is essential for BBB maintenance under inflammatory conditions.

Cortactin knockdown results in disruption of basal TBCs and alters turnover of Sertoli cell ESs in Rattus norvegicus†.

Here we explore the prediction that long-term knockdown of cortactin (CTTN), a component of tubulobulbar complexes (TBCs), disrupts TBCs in Sertoli cells and alters the turnover of basal ectoplasmic specializations (ESs). In rats, intratesticular injections of siRNA targeting CTTN (siCTTN) in one testis and nontargeting siRNA (siControl) in the contralateral testis were done on days 0, 2, 4, 6, and 8. The experiment was terminated on day 9 and testes were analyzed by either western blotting, or by stimulated emission depletion (STED), electron and/or conventional fluorescence microscopy. Levels of CTTN were successfully knocked down in experimental testes compared to controls. When cryo-sections were labeled for actin filaments, or CTTN, and oxysterol binding protein-related protein 9 (ORP9) and analyzed by STED microscopy, TBCs were "less distinct" than in tubules of the same stages from control testes. When analyzed by electron microscopy, redundant clumps of basal actin filament containing ESs were observed in experimental sections. Using labeling of actin filaments in ESs, thresholding techniques were used to calculate the number of pixels above threshold per unit length of tubule wall in seminiferous tubules at Stage VII. Median values were higher in experimental testes relative to controls in the four animals analyzed. Although we detected subtle differences in ES turnover, we were unable to demonstrate changes in spermatocyte translocation or in the levels of junction proteins at the sites. Our results are the first to demonstrate that perturbation of basal TBCs alters the turnover of actin-related junctions (ESs).

Rorqual whale nasal plugs: protecting the respiratory tract against water entry and barotrauma.

The upper respiratory tract of rorquals, lunge-feeding baleen whales, must be protected against water incursion and the risk of barotrauma at depth, where air-filled spaces like the bony nasal cavities may experience high adverse pressure gradients. We hypothesize these two disparate tasks are accomplished by paired cylindrical nasal plugs that attach on the rostrum and deep inside the nasal cavity. Here, we present evidence that the large size and deep attachment of the plugs is a compromise, allowing them to block the nasal cavities to prevent water entry while also facilitating pressure equilibration between the nasal cavities and ambient hydrostatic pressure (Pamb) at depth. We investigated nasal plug behaviour using videos of rorquals surfacing, plug morphology from dissections, histology and MRI scans, and plug function by mathematically modelling nasal pressures at depth. We found each nasal plug has three structurally distinct regions: a muscular rostral region, a predominantly fatty mid-section and an elastic tendon that attaches the plug caudally. We propose muscle contraction while surfacing pulls the fatty sections rostrally, opening the nasal cavities to air, while the elastic tendons snap the plugs back into place, sealing the cavities after breathing. At depth, we propose Pamb pushes the fatty region deeper into the nasal cavities, decreasing air volume by about half and equilibrating nasal cavity to Pamb, preventing barotrauma. The nasal plugs are a unique innovation in rorquals, which demonstrate their importance and novelty during diving, where pressure becomes as important an issue as the danger of water entry.

Listeria Membrane Protrusion Collapse: Requirement of Cyclophilin A for Listeria Cell-to-Cell Spreading.

Background: Listeria generate actin-rich tubular protrusions at the plasma membrane that propel the bacteria into neighboring cells. The precise molecular mechanisms governing the formation of these protrusions remain poorly defined. Methods: In this study, we demonstrate that the prolyl cis-trans isomerase (PPIase) cyclophilin A (CypA) is hijacked by Listeria at membrane protrusions used for cell-to-cell spreading. Results: Cyclophilin A localizes within the F-actin of these structures and is crucial for their proper formation, as cells depleted of CypA have extended actin-rich structures that are misshaped and are collapsed due to changes within the F-actin network. The lack of structural integrity within the Listeria membrane protrusions hampers the microbes from spreading from CypA null cells. Conclusions: Our results demonstrate a crucial role for CypA during Listeria infections.

Bi-directional gap junction-mediated soma-germline communication is essential for spermatogenesis.

Soma-germline interactions play conserved essential roles in regulating cell proliferation, differentiation, patterning and homeostasis in the gonad. In the Drosophila testis, secreted signalling molecules of the JAK-STAT, Hedgehog, BMP and EGF pathways are used to mediate soma-germline communication. Here, we demonstrate that gap junctions may also mediate direct, bi-directional signalling between the soma and germ line. When gap junctions between the soma and germ line are disrupted, germline differentiation is blocked and germline stem cells are not maintained. In the soma, gap junctions are required to regulate proliferation and differentiation. Localization and RNAi-mediated knockdown studies reveal that gap junctions in the fly testis are heterotypic channels containing Zpg (Inx4) and Inx2 on the germ line and the soma side, respectively. Overall, our results show that bi-directional gap junction-mediated signalling is essential to coordinate the soma and germ line to ensure proper spermatogenesis in Drosophila. Moreover, we show that stem cell maintenance and differentiation in the testis are directed by gap junction-derived cues.

Gene-Edited Human Kidney Organoids Reveal Mechanisms of Disease in Podocyte Development.

A critical event during kidney organogenesis is the differentiation of podocytes, specialized epithelial cells that filter blood plasma to form urine. Podocytes derived from human pluripotent stem cells (hPSC-podocytes) have recently been generated in nephron-like kidney organoids, but the developmental stage of these cells and their capacity to reveal disease mechanisms remains unclear. Here, we show that hPSC-podocytes phenocopy mammalian podocytes at the capillary loop stage (CLS), recapitulating key features of ultrastructure, gene expression, and mutant phenotype. hPSC-podocytes in vitro progressively establish junction-rich basal membranes (nephrin+ podocin+ ZO-1+ ) and microvillus-rich apical membranes (podocalyxin+ ), similar to CLS podocytes in vivo. Ultrastructural, biophysical, and transcriptomic analysis of podocalyxin-knockout hPSCs and derived podocytes, generated using CRISPR/Cas9, reveals defects in the assembly of microvilli and lateral spaces between developing podocytes, resulting in failed junctional migration. These defects are phenocopied in CLS glomeruli of podocalyxin-deficient mice, which cannot produce urine, thereby demonstrating that podocalyxin has a conserved and essential role in mammalian podocyte maturation. Defining the maturity of hPSC-podocytes and their capacity to reveal and recapitulate pathophysiological mechanisms establishes a powerful framework for studying human kidney disease and regeneration. Stem Cells 2017;35:2366-2378.

The caval sphincter in cetaceans and its predicted role in controlling venous flow during a dive.

A sphincter on the inferior vena cava can protect the heart of a diving mammal from overload when elevated abdominal pressures increase venous return, yet sphincters are reported incompetent or absent in some cetacean species. We previously hypothesized that abdominal pressures are elevated and pulsatile in fluking cetaceans, and that collagen is deposited on the diaphragm according to pressure levels to resist deformation. Here, we tested the hypothesis that cetaceans generating high abdominal pressures need a more robust sphincter than those generating low pressures. We examined diaphragm morphology in seven cetacean and five pinniped species. All odontocetes had morphologically similar sphincters despite large differences in collagen content, and mysticetes had muscle that could modulate caval flow. These findings do not support the hypothesis that sphincter structure correlates with abdominal pressures. To understand why a sphincter is needed, we simulated the impact of oscillating abdominal pressures on caval flow. Under low abdominal pressures, simulated flow oscillated with each downstroke. Under elevated pressures, a vascular waterfall formed, greatly smoothing flow. We hypothesize that cetaceans maintain high abdominal pressures to moderate venous return and protect the heart while fluking, and use their sphincters only during low-fluking periods when abdominal pressures are low. We suggest that pinnipeds, which do not fluke, maintain low abdominal pressures. Simulations also showed that retrograde oscillations could be transmitted upstream from the cetacean abdomen and into the extradural veins, with potentially adverse repercussions for the cerebral circulation. We propose that locomotion-generated pressures have influenced multiple aspects of the cetacean vascular system.

Discovery of a sensory organ that coordinates lunge feeding in rorqual whales.

Top ocean predators have evolved multiple solutions to the challenges of feeding in the water. At the largest scale, rorqual whales (Balaenopteridae) engulf and filter prey-laden water by lunge feeding, a strategy that is unique among vertebrates. Lunge feeding is facilitated by several morphological specializations, including bilaterally separate jaws that loosely articulate with the skull, hyper-expandable throat pleats, or ventral groove blubber, and a rigid y-shaped fibrocartilage structure branching from the chin into the ventral groove blubber. The linkages and functional coordination among these features, however, remain poorly understood. Here we report the discovery of a sensory organ embedded within the fibrous symphysis between the unfused jaws that is present in several rorqual species, at both fetal and adult stages. Vascular and nervous tissue derived from the ancestral, anterior-most tooth socket insert into this organ, which contains connective tissue and papillae suspended in a gel-like matrix. These papillae show the hallmarks of a mechanoreceptor, containing nerves and encapsulated nerve termini. Histological, anatomical and kinematic evidence indicate that this sensory organ responds to both the dynamic rotation of the jaws during mouth opening and closure, and ventral groove blubber expansion through direct mechanical linkage with the y-shaped fibrocartilage structure. Along with vibrissae on the chin, providing tactile prey sensation, this organ provides the necessary input to the brain for coordinating the initiation, modulation and end stages of engulfment, a paradigm that is consistent with unsteady hydrodynamic models and tag data from lunge-feeding rorquals. Despite the antiquity of unfused jaws in baleen whales since the late Oligocene (∼23-28 million years ago), this organ represents an evolutionary novelty for rorquals, based on its absence in all other lineages of extant baleen whales. This innovation has a fundamental role in one of the most extreme feeding methods in aquatic vertebrates, which facilitated the evolution of the largest vertebrates ever.

Ca2+ signaling machinery is present at intercellular junctions and structures associated with junction turnover in rat Sertoli cells.

The endoplasmic reticulum (ER) in Sertoli cells is a component of unique adhesion junctions (ectoplasmic specializations-ESs) and is closely associated with structures termed tubulobulbar complexes (TBCs) that internalize intercellular junctions during sperm release and during the translocation of spermatocytes through the blood-testis barrier. A role for the ER in Ca2+ regulation at ESs and TBCs has been suspected, but evidence for this function has proved elusive. Using electron microscopy, we define two new ER-plasma membrane (PM) contact sites in apical Sertoli cell processes. One of these sites occurs at TBCs where flattened lamellar cisternae of ER envelope the swollen bulb regions of the complexes, and where the gap between adjacent membranes is 12 nm. The other is at the periphery of apical processes where the gap between membranes is 13-14 nm. Using immunolocalization at the light and electron microscopic levels, we demonstrate that Ca2+ regulatory machinery is present at the ESs attached to spermatid heads, and at ER-PM contacts. Sarco/endoplasmic reticulum Ca2+-ATPase 2 (ATP2A2, SERCA2) is present at ESs; transient receptor potential channel subfamily M member 6 (TRPM6), Homer1 (HOMER1), and inositol 1,4,5-trisphosphate receptor (ITPR, IP3R) are present at ER-PM contacts associated with TBC bulbs; and stromal interacting molecule 1 (STIM1), Orai1 (ORAI1), and ATP2A2 are present at the ER-PM contacts around the margins of Sertoli cell apical processes. In Sertoli cells, the molecular machinery associated with ER generated Ca2+ fluxes is present in regions and structures directly related to junction remodeling-a process necessary for sperm release.

Controlling thoracic pressures in cetaceans during a breath-hold dive: importance of the diaphragm.

Internal pressures change throughout a cetacean's body during swimming or diving, and uneven pressures between the thoracic and abdominal compartments can affect the cardiovascular system. Pressure differentials could arise from ventral compression on each fluke downstroke or by a faster equilibration of the abdominal compartment with changing ambient ocean pressures compared with the thoracic compartment. If significant pressure differentials do develop, we would expect the morphology of the diaphragm to adapt to its in vivo loading. Here, we tested the hypothesis that significant pressure differentials develop between the thoracic and abdominal cavities in diving cetaceans by examining diaphragms from several cetacean and pinniped species. We found that: (1) regions of cetacean diaphragms possess subserosal collagen fibres that would stabilize the diaphragm against craniocaudal stretch; (2) subserosal collagen covers 5-60% of the thoracic diaphragm surface, and area correlates strongly with published values for swimming speed of each cetacean species (P<0.001); and (3) pinnipeds, which do not locomote by vertical fluking, do not possess this subserosal collagen. These results strongly suggest that this collagen is associated with loads experienced during a dive, and they support the hypothesis that diving cetaceans experience periods during which abdominal pressures significantly exceed thoracic pressures. Our results are consistent with the generation of pressure differentials by fluking and by different compartmental equilibration rates. Pressure differentials during diving would affect venous and arterial perfusion and alter transmural pressures in abdominal arteries.

Novel clathrin/actin-based endocytic machinery associated with junction turnover in the seminiferous epithelium.

Tubulobulbar complexes are elaborate clathrin/actin related structures that form at sites of intercellular attachment in the seminiferous epithelium of the mammalian testis. Here we summarize what is currently known about the morphology and molecular composition of these structures and review evidence that the structures internalize intercellular junctions both at apical sites of Sertoli cell attachment to spermatids, and at basal sites where Sertoli cells form the blood-testis barrier. We present updated models of the sperm release and spermatocyte translocation mechanisms that incorporate tubulobulbar complexes into their designs.

Morphological analysis of Francisella novicida epithelial cell infections in the absence of functional FipA.

Francisella novicida is a surrogate pathogen commonly used to study infections by the potential bioterrorism agent, Francisella tularensis. One of the primary sites of Francisella infections is the liver where >90% of infected cells are hepatocytes. It is known that once Francisella enter cells it occupies a membrane-bound compartment, the Francisella-containing vacuole (FCV), from which it rapidly escapes to replicate in the cytosol. Recent work examining the Francisella disulfide bond formation (Dsb) proteins, FipA and FipB, have demonstrated that these proteins are important during the Francisella infection process; however, details as to how the infections are altered in epithelial cells have remained elusive. To identify the stage of the infections where these Dsbs might act during epithelial infections, we exploited a hepatocyte F. novicida infection model that we recently developed. We found that F. novicida ΔfipA-infected hepatocytes contained bacteria clustered within lysosome-associated membrane protein 1-positive FCVs, suggesting that FipA is involved in the escape of F. novicida from its vacuole. Our morphological evidence provides a tangible link as to how Dsb FipA can influence Francisella infections.

An Alternative Model of Tubulobulbar Complex Internalization During Junction Remodeling in the Seminiferous Epithelium of the Rat Testis.

Tubulobulbar complexes (TBCs) are elongate subcellular machines responsible for internalizing intercellular junctions during sperm release. Each complex consists of a double-membrane tubular core terminating in a clathrin-coated pit. The core is surrounded by a network of actin filaments, and a distinct swelling or bulb, which lacks an association with actin, develops in the distal third of the structure. The bulb eventually buds from the complex and enters endocytic compartments of the Sertoli cell. The relationship of the actin cuff to the formation and budding of the bulb is not known. To gain insight into this relationship, we perturbed the actin networks of TBCs with cytochalasin D. When isolated testes were perfused with a physiological buffer containing cytochalasin D, apical TBCs at stage VII of spermatogenesis were associated with lower levels of actin compared to controls. At the ultrastructural level, the actin networks in cytochalasin D-treated testes appeared patchy, and ectopic bulbs and swollen tubular regions occurred. When normal untreated samples at early stage VII were analyzed, large elongate bulbs and short tubular sections were observed. Together, these results suggest a new model for TBC vesiculation in which the actin network begins to disassemble and the tubular region begins to swell into a bulb. As actin disassembly continues, the coated pit and most of the tubular region are incorporated into the enlarging bulb. The remaining short neck of the bulb near the base of the complex undergoes scission, and the bulb is internalized.

PPP1CC2 can form a kinase/phosphatase complex with the testis-specific proteins TSSK1 and TSKS in the mouse testis.

The mouse protein phosphatase gene Ppp1cc is essential for male fertility, with mutants displaying a failure in spermatogenesis including a widespread loss of post-meiotic germ cells and abnormalities in the mitochondrial sheath. This phenotype is hypothesized to be responsible for the loss of the testis-specific isoform PPP1CC2. To identify PPP1CC2-interacting proteins with a function in spermatogenesis, we carried out GST pull-down assays in mouse testis lysates. Amongst the identified candidate interactors was the testis-specific protein kinase TSSK1, which is also essential for male fertility. Subsequent interaction experiments confirmed the capability of PPP1CC2 to form a complex with TSSK1 mediated by the direct interaction of each with the kinase substrate protein TSKS. Interaction between PPP1CC2 and TSKS is mediated through an RVxF docking motif on the TSKS surface. Phosphoproteomic analysis of the mouse testis identified a novel serine phosphorylation site within the TSKS RVxF motif that appears to negatively regulate binding to PPP1CC2. Immunohistochemical analysis of TSSK1 and TSKS in the Ppp1cc mutant testis showed reduced accumulation to distinct cytoplasmic foci and other abnormalities in their distribution consistent with the loss of germ cells and seminiferous tubule disorganization observed in the Ppp1cc mutant phenotype. A comparison of Ppp1cc and Tssk1/2 knockout phenotypes via electron microscopy revealed similar abnormalities in the morphology of the mitochondrial sheath. These data demonstrate a novel kinase/phosphatase complex in the testis that could play a critical role in the completion of spermatogenesis.

Morphology and Mechanics of the Fin Whale Esophagus: The Key to Fast Processing of Large Food Volumes by Rorquals.

Lunge feeding rorqual whales feed by engulfing a volume of prey laden water that can be as large as their own body. Multiple feeding lunges occur during a single foraging dive and the time between each lunge can be as short as 30 s (Goldbogen et al. 2013). During this short inter-lunge time, water is filtered out through baleen to concentrate prey in the oral cavity, and then the prey is swallowed prior to initiating the next lunge. Prey density in the ocean varies greatly, and despite the potential of swallowing a massive volume of concentrated prey as a slurry, the esophagus of rorqual whales has been anecdotally described as unexpectedly narrow with a limited capacity to expand. How rorquals swallow large quantities of food down a narrow esophagus during a limited inter-lunge time remains unknown. Here, we show that the small diameter muscular esophagus in the fin whale is optimized to transport a slurry of food to the stomach. A thick wall of striated muscle occurs at the pharyngeal end of the esophagus which, together with the muscular wall of the pharynx, may generate a pressure head for transporting the food down the esophagus to the stomach as a continuous stream rather than separating the food into individual boluses swallowed separately. This simple model is consistent with estimates of prey density and stomach capacity. Rorquals may be the only animals that capture a volume of food too large to swallow as a single intact bolus without oral processing, so the adaptations of the esophagus are imperative for transporting these large volumes of concentrated food to the stomach during a time-limited dive involving multiple lunges.

Myosin VI Is Associated With the Endoplasmic Reticulum in Regions of Sertoli Cells Containing Tubulobulbar Complexes.

Myosin VI has been reported by others to localize in association with various regions of apical tubulobulbar complexes (TBCs) at sites of attachment between Sertoli cells and late spermatids in the mouse. Tubulobulbar complexes internalize "intact" intercellular junctions during sperm release and during spermatocyte translocation through the blood-testis barrier. Here, we use super-resolution (STED-stimulated emission depletion) and electron microscopy of immunolabeled sections of rat testis to clearly define the localization of anti-myosin VI reactivity both at apical and basal sites in the epithelium. In data stacks collected by STED imaging, staining at TBCs was predominantly associated with bulb regions of the complexes. At apical sites, when data stacks were analyzed with an Imaris software, staining appeared around and extended between adjacent bulbs. At basal sites, in addition to labeling at TBC bulbs, reactive sites appeared concentrated in regions close to but not directly associated with intercellular junctions. At the ultrastructural level, labeling was predominantly associated with cisternae of the endoplasmic reticulum associated with the bulbs of TBCs and near to basal junction complexes. We conclude that myosin VI may be associated with specific subdomains of the endoplasmic reticulum related to TBC bulbs and associated basal junction complexes between Sertoli cells.

The Soft Palate Enables Extreme Feeding and Explosive Breathing in the Fin Whale (Balaenoptera physalus).

The evolution of lunge feeding in rorqual whales was associated with the evolution of several unique morphological features that include non-synovial ligamentous temporomandibular joints, a tongue that can invert and extend backward to the umbilicus, walls of the oral cavity that can dramatically expand, and muscles and nerves that are stretchy. Also, among the acquired features was an enlargement of the rostral end of the soft palate into an oral plug that occludes the opening between the oral cavity and pharynx and prevents water incursion into the pharynx during the engulfment phase of a feeding lunge. During this engulfment phase of a lunge, the volume of water entering the oral cavity can exceed the volume of the whale itself. Here, using dissection of fetuses and adults and a magnetic resonance imaging dataset of a fetus, we examine the detailed anatomy of the soft palate in fin whales. We describe several innovative features relative to other mammals, including changes in the attachment and positions of the major extrinsic muscles of the palate, alterations in the morphology of the pterygoid processes related to the palate and pharynx, and the presence of distinct muscle layers in the part of the palate caudal to the oral plug. Based on the anatomy, we present a model for how the soft palate is positioned at rest, and how it functions during feeding, breathing, and swallowing.

Videofluoroscopy of the aerodigestive tract in Phoca vitulina: reshaping perspectives on translational medicine.

Thousands of rescued harbor seals (Phoca vitulina) require rehabilitation worldwide. Many require resource intensive gavage feeding due to abandonment soon after birth. Little is known about seal swallowing, therefore, our primary objective was to determine the feasibility of conducting videofluoroscopic swallowing studies (VFS) on seal pups prior to their release. Secondarily, we propose swallowing phase descriptions. We adapted a VFS approach used in humans and our feasibility parameters included: bolus detection and consumption, and number of analyzable swallowing events. Unrestrained seals were imaged in a dry environment using a Siemens mobile c-arm fluoroscopy unit. Oral boluses were thawed herring injected with liquid barium suspension (105% w/v). Two independent raters described swallows using a standardized approach with results summarized descriptively. We successfully completed freely-behaving VFS with two infant seals (1 male: 8 wks, 3 d; 1 female: 5 wks, 3 d). Both consumed five boluses with six fully analyzable swallowing events. We describe four swallow phases: preparatory, prehension, oropharyngeal and esophageal. Airway protection likely occurs in two ways: (1) during the preparatory phase through modified corniculate cartilage contact with the glottis and (2) with soft palate contact to the base of tongue prior to swallow initiation. We have conducted a unique VFS approach on rehabilitated seals, prior to their release. We have described airway protection and suggest that swallowing is initiated earlier in the feeding process than described previously. This protocol success will afford: (1) collection of normative swallowing data, and (2) future knowledge translation from humans to seals.

Membrane contact site detection (MCS-DETECT) reveals dual control of rough mitochondria-ER contacts.

Identification and morphological analysis of mitochondria-ER contacts (MERCs) by fluorescent microscopy is limited by subpixel resolution interorganelle distances. Here, the membrane contact site (MCS) detection algorithm, MCS-DETECT, reconstructs subpixel resolution MERCs from 3D super-resolution image volumes. MCS-DETECT shows that elongated ribosome-studded riboMERCs, present in HT-1080 but not COS-7 cells, are morphologically distinct from smaller smooth contacts and larger contacts induced by mitochondria-ER linker expression in COS-7 cells. RiboMERC formation is associated with increased mitochondrial potential, reduced in Gp78 knockout HT-1080 cells and induced by Gp78 ubiquitin ligase activity in COS-7 and HeLa cells. Knockdown of riboMERC tether RRBP1 eliminates riboMERCs in both wild-type and Gp78 knockout HT-1080 cells. By MCS-DETECT, Gp78-dependent riboMERCs present complex tubular shapes that intercalate between and contact multiple mitochondria. MCS-DETECT of 3D whole-cell super-resolution image volumes, therefore, identifies novel dual control of tubular riboMERCs, whose formation is dependent on RRBP1 and size modulated by Gp78 E3 ubiquitin ligase activity.