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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). Summary sentence Cortactin knockdown disrupts basal TBCs and perturbs basal ES turnover. Graphical Abstract

Identification and morphological analysis of mitochondria-ER contacts (MERCs) by fluorescent microscopy is limited by sub-pixel resolution inter-organelle distances. Application of a Membrane Contact Site (MCS) detection algorithm, MCS-DETECT, to 3D STED super-resolution image volumes reconstructs sub-resolution MERCs. 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 expression is reduced in Gp78 knockout HT-1080 cells and induced by Gp78 ubiquitin ligase activity in COS-7 cells. Knockdown of the 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, that are lost upon RRBP1 knockdown. MCS-DETECT of 3D whole cell super-resolution image volumes therefore identifies a novel dual regulatory mechanism for tubular riboMERCs, whose formation is dependent on RRBP1 and size modulated by Gp78 E3 ubiquitin ligase activity.eTOC SummaryApplication of the sub-pixel resolution Membrane Contact Site (MCS) detection algorithm, MCS-DETECT, to 3D STED super-resolution image volumes identifies a novel dual regulatory mechanism for tubular riboMERCs, whose formation is dependent on RRBP1 and size modulated by Gp78 E3 ubiquitin ligase activity.

Abandoned harbor seal pups (Phoca vitulina) are frequently recovered by rehabilitation centers and often require intensive nursing, gavage feeding and swallowing rehabilitation prior to anticipated release. Seal upper aerodigestive tract (UAT) histology descriptions relevant to deglutition are limited, impacting advances in rehabilitation practice. Therefore, we examined the histological characteristics of the harbor seal UAT to understand species-specific functional anatomy and characterize adaptations. To this end, we conducted gross dissections, compiled measurements and reviewed histologic features of the UAT structures of 14 preweaned harbor seal pups that died due to natural causes or were humanely euthanized. Representative samples for histologic evaluation included the tongue, salivary glands, epiglottis, and varying levels of the trachea and esophagus. Histologically, there was a prominent muscularis in the tongue with fewer lingual papillae types compared to humans. Abundant submucosal glands were observed in lateral and pharyngeal parts of the tongue and rostral parts of the esophagus. When compared to other mammalian species, there was a disproportionate increase in the amount of striated muscle throughout the length of the esophageal muscularis externa. This may indicate a lesser degree of autonomic control over the esophageal phase of swallowing in harbor seals. Our study represents the first detailed UAT histological descriptions for neonatal harbor seals. Collectively, these findings support specific anatomic and biomechanical adaptations relevant to suckling, prehension, and deglutition. This work will inform rehabilitation practices and guide future studies on swallowing physiology in harbor seals with potential applications to other pinniped and otariid species in rehabilitation settings.

The release of late spermatids from the seminiferous epithelium requires the internalization of intercellular junctions by Sertoli cell specific structures called “tubulobulbar complexes” (TBCs). These large, endocytic devices likely evolved from classic clathrin-mediated-endocytosis (CME) machinery, but have several important morphological differences to CME vesicles. Most notable among these differences is that extensive endoplasmic reticulum (ER) membrane contact sites (MCSs) occur with TBCs and not with clathrin-coated pits. One of the well-established functions of ER MCSs is lipid exchange. Previously, we have established that the ORP9 lipid exchange protein is localized to the TBC-ER MCS; however, the function of ORP9 and lipid exchange at the sites is not known. Here we use an in vivo knockdown approach to probe function. The testes of Sprague–Dawley rats were injected with ORP9 targeted siRNA or non-targeted reagents, and the tissues examined by bright field, super-resolution stimulated emission depletion, and electron microscopy. The knockdown of ORP9 was achieved and maintained with daily injections of siRNA for 2-3 day intervals. Compared to controls, sections from ORP9 siRNA-injected testes had longer TBC tubes and fewer fused TBC bulbs. Late spermatids were also abnormally retained in the epithelium of knockdown tissue. These results suggest that ORP9 is necessary for normal TBC bulb vesiculation and fusion, most likely by changing the plasma membrane lipid profile of the TBC. These data also further support the conclusion that TBCs are part of the normal mechanism of sperm release.

Dominant and recessive mutations in podocalyxin (PODXL) are associated with human kidney disease. Interestingly, some PODXL mutations manifest as anuria while others are associated with proteinuric kidney disease. PODXL heterozygosity is associated with adult-onset kidney disease and podocalyxin shedding into the urine is a common biomarker of a variety nephrotic syndromes. It is unknown, however, how various lesions in PODXL contribute to these disparate disease pathologies. Here we generated two mouse stains: one that deletes Podxl in developmentally mature podocytes (Podxl∆Pod) and a second that is heterozygous for podocalyxin in all tissues (Podxl+/−). We used histologic and ultrastructural analyses, as well as clinical chemistry assays to evaluate kidney development and function in these strains. In contrast to null knockout mice (Podxl−/−), which die shortly after birth from anuria and hypertension, Podxl∆Pod mice develop an acute congenital nephrotic syndrome characterized by focal segmental glomerulosclerosis (FSGS) and proteinuria. Podxl+/− mice, in contrast, have a normal lifespan, and fail to develop kidney disease under normal conditions. Intriguingly, although wild-type C57Bl/6 mice are resistant to puromycin aminonucleoside (PA)-induced nephrosis (PAN), Podxl+/− mice are highly sensitive and PA induces severe proteinuria and collapsing FSGS. In summary, we find that the developmental timepoint at which podocalyxin is ablated (immature vs. mature podocytes) has a profound effect on the urinary phenotype due to its critical roles in both the formation and the maintenance of podocyte ultrastructure. In addition, Podxl∆Pod and Podxl+/− mice offer powerful new mouse models to evaluate early biomarkers of proteinuric kidney disease and to test novel therapeutics.

Background Ulcerative colitis (UC) is a chronic inflammatory condition linked to intestinal microbial dysbiosis, including the expansion of E. coli strains related to extra-intestinal pathogenic E. coli. These “pathobionts” exhibit pathogenic properties, but their potential to promote UC is unclear due to the lack of relevant animal models. Aims We explored the potential to establish a mouse model of GI infection by the UC-associated E. coli strain p19A, as well as characterize the pathogenic features of p19A. Methods We used a representative UC pathobiont strain (p19A), and mice lacking single immunoglobulin and toll-interleukin 1 receptor domain (SIGIRR), a deficiency increasing susceptibility to gut infections. Vancomycin-pretreated Sigirr-/- mice were subsequently gavaged with the control E. coli DH10B (a derivative of commensal strain K-12) or p19A. One day after infection, mice were exposed to 2.5% dextran sodium sulfate (DSS) in their drinking water for another 4 days. Results Strain p19A was found to adhere to the cecal mucosa of Sigirr-/- mice, causing modest inflammation. Moreover, it dramatically worsened DSS-induced colitis. This potentiation was attenuated using a p19A strain lacking α-hemolysin genes, or when we targeted pathobiont adherence using a p19A strain lacking the adhesin FimH, or following treatment with FimH antagonists. Conclusions Thus, UC pathobionts adhere to the intestinal mucosa, and worsen the course of colitis in susceptible hosts in a manner dependent on specific virulence factors, including α-hemolysin and FimH. Funding Agencies CCC, CIHR

Separation of respiratory and digestive tracts in the mammalian pharynx is critical for survival. Food must be kept out of the respiratory tract, and air must be directed into the respiratory tract when breathing.

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 (Podxl(ΔTie2Cre)). Although we did not detect substantially altered permeability in naïve mice, systemic priming with lipopolysaccharide (LPS) selectively disrupted the blood–brain barrier (BBB) in Podxl(ΔTie2Cre) mice. To study the potential consequence of this BBB breach, we used a selective agonist (TFLLR-NH(2)) 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-NH(2), LPS-primed Podxl(ΔTie2Cre) 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.

The actin cytoskeleton has long been recognized as a crucial sub-cellular filament system that is responsible for governing fundamental events ranging from cell division and muscle contraction to whole cell motility and the maintenance of tissue integrity. Consequently, it is not surprising that this network is the focus of over 100,000 different manuscripts. Alterations in the actin cytoskeleton lead to an assortment of diseases and serve as a target for a variety of pathogens. Here we have brought together a collection of primary research articles and reviews that underscore the broad influence this filament system has on organisms. Anat Rec, 301:1986-1990, 2018. © 2018 Wiley Periodicals, Inc.

Direct cell-to-cell spreading of Listeria monocytogenes requires the bacteria to induce actin-based finger-like membrane protrusions in donor host cells that are endocytosed through caveolin-rich membrane invaginations by adjacent receiving cells. An actin shell surrounds these endocytic sites; however, its structure, composition, and functional significance remain elusive. Here, we show that the formin mDia1, but surprisingly not the Arp2/3 complex, is enriched at the membrane invaginations generated by L. monocytogenes during HeLa and Jeg-3 cell infections. Electron microscopy reveals a band of linear actin filaments that run along the longitudinal axis of the invagination membrane. Mechanistically, mDia1 expression is vital for the assembly of this F-actin shell. mDia1 is also required for the recruitment of Filamin A, a caveola-associated F-actin cross-linking protein, and caveolin-1 to the invaginations. Importantly, mixed-cell infection assays show that optimal caveolin-based L. monocytogenes cell-to-cell spreading correlates with the formation of the linear actin filament-containing shell by mDia1. IMPORTANCE Listeria monocytogenes spreads from one cell to another to colonize tissues. This cell-to-cell movement requires the propulsive force of an actin-rich comet tail behind the advancing bacterium, which ultimately distends the host plasma membrane into a slender bacterium-containing membrane protrusion. These membrane protrusions induce a corresponding invagination in the membrane of the adjacent host cell. The host cell that receives the protrusion utilizes caveolin-based endocytosis to internalize the structures, and filamentous actin lines these membrane invaginations. Here, we set out to determine the structure and function of this filamentous actin “shell.” We demonstrate that the formin mDia1, but not the Arp2/3 complex, localizes to the invaginations. Morphologically, we show that this actin is organized into linear arrays and not branched dendritic networks. Mechanistically, we show that the actin shell is assembled by mDia1 and that mDia1 is required for efficient cell-to-cell transfer of L. monocytogenes.

Effective 'valving' in the upper aerodigestive tract (UAT) is essential to temporarily separate the digestive and respiratory pathways. Marine mammals are largely dedicated to feeding underwater, and in many cases swallowing prey whole. In seals, little work has been done to explore the anatomy and function of the UAT in the context of valving mechanisms that function to separate food and air pathways. Here we use videofluoroscopy, gross dissection, histology and computed tomography (CT) renderings to explore the anatomy of the larynx and soft palate in the harbour seal (

Prestin is an integral membrane motor protein located in outer hair cells of the mammalian cochlea. It is responsible for electromotility and required for cochlear amplification. Although prestin works in a cycle-by-cycle mode up to frequencies of at least 79 kHz, it is not known whether or not prestin is required for the extreme high frequencies used by echolocating species. Cetaceans are known to possess a prestin coding gene. However, the expression and distribution pattern of the protein in the cetacean cochlea has not been determined, and the contribution of prestin to echolocation has not yet been resolved. Here we report the expression of the protein prestin in five species of echolocating whales and two species of echolocating bats. Positive labeling in the basolateral membrane of outer hair cells, using three anti-prestin antibodies, was found all along the cochlear spiral in echolocating species. These findings provide morphological evidence that prestin can have a role in cochlear amplification in the basolateral membrane up to 120–180 kHz. In addition, labeling of the cochlea with a combination of anti-prestin, anti-neurofilament, anti-myosin VI and/or phalloidin and DAPI will be useful for detecting potential recent cases of noise-induced hearing loss in stranded cetaceans. This study improves our understanding of the mechanisms involved in sound transduction in echolocating mammals, as well as describing an optimized methodology for detecting cases of hearing loss in stranded marine mammals.

Tubulobulbar complexes (TBCs) internalize intercellular junctions during sperm release. One of the characteristic features of TBCs is that they form “bulbs” or swollen regions that have well-defined membrane contact sites (MCS) with adjacent cisternae of endoplasmic reticulum. Previously, we have localized the IP3R calcium channel to the TBC bulb-ER contacts and have hypothesized that fluctuations in local calcium levels may facilitate the maturation of TBC bulbs into putative endosomes, or alter local actin networks that cuff adjacent tubular regions of the TBCs. To test this, we injected the testes of Sprague Dawley rats with small interfering RNAs (siRNAs) against IP3R1 and processed the tissues for either western blot, immunofluorescence, or electron microscopy. When compared to control testes injected with nontargeting siRNAs, Sertoli cells in knocked-down testes showed significant morphological alterations to the actin networks including a loss of TBC actin and the appearance of ectopic para-crystalline actin bundles in Sertoli cell stalks. There also was a change in the abundance and distribution of TBC-ER contact sites and large internalized endosomes. This disruption of TBCs resulted in delay of the withdrawal of apical processes away from spermatids and in spermiation. Together, these findings are consistent with the hypothesis that calcium exchange at TBC-ER contacts is involved both in regulating actin dynamics at TBCs and in the maturing of TBC bulbs into endosomes. The results are also consistent with the hypothesis that TBCs are part of the sperm release mechanism.

Listeria monocytogenes moves from one cell to another as it disseminates within tissues. This bacterial transfer process depends on the host actin cytoskeleton as the bacterium forms motile actin-rich membranous protrusions that propel the bacteria into neighboring cells, thus forming corresponding membrane invaginations. Here, we examine these membrane invaginations and demonstrate that caveolin-1–based endocytosis is crucial for efficient bacterial cell-to-cell spreading. We show that only a subset of caveolin-associated proteins (cavin-2 and EHD2) are involved in this process. Despite the absence of clathrin at the invaginations, the classical clathrin-associated protein epsin-1 is also required for efficient bacterial spreading. Using isolated L. monocytogenes protrusions added onto naive host cells, we demonstrate that actin-based propulsion is dispensable for caveolin-1 endocytosis as the presence of the protrusion/invagination interaction alone triggers caveolin-1 recruitment in the recipient cells. Finally, we provide a model of how this caveolin-1–based internalization event can exceed the theoretical size limit for this endocytic pathway., Listeria monocytogenes moves from one cell to another using actin-rich membrane protrusions that propel the bacterium toward neighboring cells. Despite cholesterol being required for this transfer process, the precise host internalization mechanism remains elusive. Here, we show that caveolin endocytosis is key to this event as bacterial cell-to-cell transfer is severely impaired when cells are depleted of caveolin-1. Only a subset of additional caveolar components (cavin-2 and EHD2) are present at sites of bacterial transfer, and although clathrin and the clathrin-associated proteins Eps15 and AP2 are absent from the bacterial invaginations, efficient L. monocytogenes spreading requires the clathrin-interacting protein epsin-1. We also directly demonstrated that isolated L. monocytogenes membrane protrusions can trigger the recruitment of caveolar proteins in a neighboring cell. The engulfment of these bacterial and cytoskeletal structures through a caveolin-based mechanism demonstrates that the classical nanometer-scale theoretical size limit for this internalization pathway is exceeded by these bacterial pathogens.

Effective ‘valving’ in the upper aerodigestive tract (UAT) is essential to temporarily separate the digestive and respiratory pathways. Marine mammals are largely dedicated to feeding underwater, and in many cases swallowing prey whole. In seals, little work has been done to explore the anatomy and function of the upper aerodigestive tract in the context of valving mechanisms that function to separate food and air pathways. Here we use videofluoroscopy, gross dissection, histology and CT renderings to explore the anatomy of the larynx and soft palate in the harbour seal (Phoca vitulina), and generate models for how valving mechanisms in the head and neck may function during breathing, phonating, diving and swallowing. Harbour seals have an elevated larynx and the epiglottis may rise above the level of the soft palate, particularly in pups when sucking. In addition, the corniculate and arytenoid cartilages with associated muscles form most of the lateral margins of the laryngeal inlet and vestibule, and move independently to facilitate airway closure. The corniculate cartilages flex over the laryngeal inlet beneath the epiglottis to completely close the laryngeal vestibule and inlet. The vocal folds are thick and muscular and the medial margin of the folds contains a small vocal ligament. The soft palate has well-defined levator veli palatini muscles that likely function to elevate the palate and close the pharyngeal isthmus during feeding. Our results support the conclusion that harbour seals have evolved UAT valving mechanisms as adaptations to a marine environment that are not seen in terrestrial carnivores.

The upper respiratory tract of rorquals, lunge-feeding baleen whales, must be protected against water incursion and against 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 that demonstrate their importance and novelty during diving, where pressure becomes as important an issue as the danger of water entry.

Paneth cells are a key subset of secretory epithelial cells found at the base of small intestinal crypts. Unlike intestinal goblet cells, which secrete the mucin Muc2, Paneth cells are best known for producing an array of antimicrobial factors. We unexpectedly identified Muc2 staining localized around Paneth cell granules. Electron microscopy (EM) confirmed an electron lucent halo around these granules, which was lost in Paneth cells from Muc2-deficient (−/−) mice. EM and immunostaining for lysozyme revealed that Muc2−/− Paneth cells contained larger, more densely packed granules within their cytoplasm, and we detected defects in the transcription of key antimicrobial genes in the ileal tissues of Muc2−/− mice. Enteroids derived from the small intestine of wild-type and Muc2−/− mice revealed phenotypic differences in Paneth cells similar to those seen in vivo. Moreover, lysozyme-containing granule release from Muc2−/− enteroid Paneth cells was shown to be impaired. Surprisingly, Paneth cells within human ileal and duodenal tissues were found to be Muc2 negative. Thus Muc2 plays an important role in murine Paneth cells, suggesting links in function with goblet cells; however human Paneth cells lack Muc2, highlighting that caution should be applied when linking murine to human Paneth cell functions. NEW & NOTEWORTHY We demonstrate for the first time that murine Paneth cell granules possess a halo comprised of the mucin Muc2. The presence of Muc2 exerts an impact on Paneth cell granule size and number and facilitates the release and dispersal of antimicrobials into the mucus layer. Interestingly, despite the importance of Muc2 in murine Paneth cell function, our analysis of Muc2 in human intestinal tissues revealed no trace of Muc2 expression by human Paneth cells.

In this commentary, I provide an introduction to and the context for the four articles in the thematic series published to celebrate the Editorial Board Meeting of the Anatomical Record in Vancouver, British Columbia, Canada in July of 2017. The articles describe various aspects of whale anatomy and the potential for a new generation of digital tags to provide information on functional anatomy of free swimming animals in the wild. The whales described are all native to the northwest coast of North America, as well as being found elsewhere, and the authors are related in some way to the University of British Columbia in Vancouver. Anat Rec, 300:1930-1934, 2017. © 2017 Wiley Periodicals, Inc.

Nerves that supply the floor of the oral cavity in rorqual whales are extensible to accommodate the dramatic changes in tissue dimensions that occur during "lunge feeding" in this group. We report here that the large nerves innervating the muscle component of the ventral grooved blubber (VGB) in fin whales are branches of cranial nerve VII (facial nerve). Therefore, the muscles of the VGB are homologous to second branchial arch derived muscles, which in humans include the muscles of "facial expression." We speculate, based on the presence of numerous foramina on the dorsolateral surface of the mandibular bones, that general sensation from the VGB likely is carried by branches of the mandibular division (V3) of cranial nerve V (trigeminal nerve), and that these small branches travel in the lipid-rich layer directly underlying the skin. We show that intercostal and phrenic nerves, which are not extensible, have a different wall and nerve core morphology than the large VGB nerves that are branches of VII. Although these VGB nerves are known to have two levels of waviness, the intercostal and phrenic nerves have only one in which the nerve fascicles in the nerve core are moderately wavy. In addition, the VGB nerves have inner and outer parts to their walls with numerous large elastin fibers in the outer part, whereas intercostal and phrenic nerves have single walls formed predominantly of collagen. Our results illustrate that overall nerve morphology depends greatly on location and the forces to which the structures are exposed. Anat Rec, 300:1963-1972, 2017. © 2017 Wiley Periodicals, Inc.

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.

Tubulobulbar complexes are clathrin/actin-based structures that internalize intercellular junctions in the testis. They resemble coated pits with extremely long necks that are cuffed by dendritic actin networks. As the structures mature, a swollen region or bulb develops near the end of each complex. The bulbs lack actin cuffs and are closely associated with cisternae of endoplasmic reticulum. The bulbs expand and are internalized and enter endocytic compartments of the Sertoli cell. Previous immunofluorescence studies have demonstrated that markers for early endosomes (Rab5 and EEA1) are associated with tubulobulbar complexes and are localized at or near the ends of the structures. Here we use a pre-embedding immunoelectron microscopic technique to accurately localize these markers to apical tubulobulbar complexes that occur at junctions between Sertoli cells and spermatids. Staining for Rab5 occurs at bulbs, identified by the presence of two plasma membranes and a close association with cisternae of endoplasmic reticulum. EEA1 is associated with large vesicles that lack an association with the endoplasmic reticulum. Labeling for nectin-3, an adhesion junction protein in the spermatid plasma membrane, occurs at junctions, TBC bulbs, and in associated double membrane vesicles. Our results suggest that Rab5 associates with junction protein containing bulbs prior to their internalization and that EEA1 associates with the structures later and after internalization. We conclude that at tubulobulbar complexes in Sertoli cells of the seminiferous epithelium, the identity of 'bulbs' as putative early endosomes begins to be established prior to their undergoing scission or budding from their parent structures. Anat Rec, 300:1160-1170, 2017. © 2017 Wiley Periodicals, Inc.

Basal tubulobulbar complexes (TBCs) that occur at attachment sites between neighboring Sertoli cells are subcellular machines that internalize intercellular junctions during movement of spermatocytes from basal to adluminal compartments of the seminiferous epithelium. Each complex consists of an elongate tubular extension of two attached plasma membranes, and is capped at its distal end by a clathrin-coated pit. The tubular region is surrounded by a cuff of actin arranged in a dendritic network. Near the end of the complex, a bulbous region forms that lacks the actin cuff but is closely associated with cisternae of endoplasmic reticulum. The bulb eventually buds from the complex and enters endocytic compartments of the Sertoli cell. Previous research has shown that when the actin network is perturbed using the actin filament-disruptor, cytochalasin D, apical tubulobulbar complexes that are associated with spermatids were associated with lower levels of actin, patchy actin networks and swollen tubular regions. Here we explored the effects of actin network perturbation on the morphology of basal tubulobulbar complexes in stage V seminiferous tubules. Isolated rat testes were perfused ex vivo for one hour with oxygenated Krebs-Henseleit buffer (with BSA) containing either 40 μM cytochalasin D or control solution containing DMSO and perfusion-fixed for electron microscopy. Compared to control, actin cuffs in drug-treated TBCs appeared less uniform and patchy. In addition, the tubular regions of the complexes appeared swollen. Our results are consistent with the conclusion that intact networks of actin filaments are required for maintaining the structural integrity of basal TBCs. Anat Rec, 299:1449-1455, 2016. © 2016 Wiley Periodicals, Inc.

Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro . Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin- or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated (in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility. IMPORTANCE Structures containing branched actin filaments require the Arp2/3 complex. One of the most commonly used systems to study intracellular movement generated by Arp2/3-based actin motility exploits actin-rich comet tails made by Listeria . Using these infections together with live imaging and cell-free protein reconstitution experiments, we show that another protein, palladin, can be used in place of Arp2/3 to form actin-rich structures. Additionally, we show that palladin is needed for the structural integrity of comet tails as its depletion or mutation of critical regions causes dramatic changes to comet tail organization. These findings are the first to identify a protein that can functionally replace the Arp2/3 complex and have implications for all actin-based structures thought to exclusively use that complex.

Sertoli cells of the mammalian seminiferous epithelium form unique subcellular actin-related structures at intercellular junctions. The appearance of these so called "tubulobulbar complexes" (TBCs) precedes both sperm release at the apex of the epithelium and the movement of early spermatogenic cells out of the spermatogonial stem cell niche at the base of the epithelium. TBCs are considered to be part of the mechanism of junction endocytosis by Sertoli cells. The structures contain junction proteins and morphologically identifiable junctions, and are associated with markers of endocytosis. Here we review the current state of knowledge about the structure and function of TBCs. As the complexes form, they morphologically resemble and have the molecular signature of clathrin-coated pits with extremely long necks. As they mature, the actin filament networks around the "necks" of the structures progressively disassemble and the membrane cores expand or swell into distinct "bulbs". These bulbs acquire extensive membrane contact sites with associated cisternae of endoplasmic reticulum. Eventually the bulbs undergo scission and continue through endosomal compartments of the Sertoli cells. The morphology and composition of TBC indicates to us that the structures likely evolved from the basic clathrin-mediated endocytosis mechanism common to cells generally, and along the way they incorporated unique features to accommodate the cyclic turnover of massive and "intact" intercellular junctions that occurs during spermatogenesis. Anat Rec, 301:2080-2085, 2018. © 2018 Wiley Periodicals, Inc.

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 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 pinnipeds, which do not fluke, maintain low abdominal pressures. Simulations also showed 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.

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.

Whales are important model systems for understanding the physiological and ecological consequences of extreme body size. However, whales are also some of the most difficult animals to study because their large size precludes experimental studies under controlled conditions. Here we review a wide range of morphological studies that enable greater inference of physiological processes. In particular, we focus on baleen whales that exhibit extensive diving and foraging adaptations. Using morphological data, we (i) explore the biomechanics and sensory physiology of lunge-feeding rorqual whales (Balaenopteridae), (ii) determine the effects of scale and diving pressures on the circulatory physiology of fin whales (Balaenoptera physalus (L., 1758)), and (iii) better understand the adaptations of the cetacean respiratory system that facilitate a fully aquatic life history. These studies underscore the value of understanding functional morphology in animals that cannot be studied using traditional laboratory techniques.

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.