Large Multivesicular Bodies Research Articles

Chloride Channel-3 (ClC-3) Modifies the Trafficking of Leucine-Rich Repeat-Containing 8A (LRRC8A) Anion Channels.

Chloride channel-3 (ClC-3) Cl-/H+ antiporters and leucine-rich repeat-containing 8 (LRRC8) family anion channels have both been associated with volume-regulated anion currents (VRACs). VRACs are often altered in ClC-3 null cells but are absent in LRRC8A null cells. To explore the relationship between ClC-3, LRRC8A, and VRAC we localized tagged proteins in human epithelial kidney (HEK293) cells using multimodal microscopy. Expression of ClC-3-GFP induced large multivesicular bodies (MVBs) with ClC-3 in the delimiting membrane. LRRC8A-RFP localized to the plasma membrane and to small cytoplasmic vesicles. Co-expression demonstrated co-localization in small, highly mobile cytoplasmic vesicles that associated with the early endosomal marker Rab5A. However, most of the small LRRC8A-positive vesicles were constrained within large MVBs with abundant ClC-3 in the delimiting membrane. Dominant negative (S34A) Rab5A prevented ClC-3 overexpression from creating enlarged MVBs, while constitutively active (Q79L) Rab5A enhanced this phenotype. Thus, ClC-3 and LRRC8A are endocytosed together but independently sorted in Rab5A MVBs. Subsequently, LRRC8A-labeled vesicles were sorted to MVBs labeled by Rab27A and B exosomal compartment markers, but not to Rab11 recycling endosomes. VRAC currents were significantly larger in ClC-3 null HEK293 cells. This work demonstrates dependence of LRRC8A trafficking on ClC-3 which may explain the association between ClC-3 and VRACs.

The phospholipid flippase ATP8B1 is required for lysosomal fusion in macrophages.

ATP8B1 is a phospholipid flippase and member of the type 4 subfamily of P-type ATPases(P4-ATPase) subfamily. P4-ATPases catalyze the translocation of phospholipids across biological membranes, ensuring proper membrane asymmetry, which is crucial for membrane protein targeting and activity, vesicle biogenesis, and barrier function. Here we have investigated the role of ATP8B1 in the endolysosomal pathway in macrophages. Depletion of ATP8B1 led to delayed degradation of content in the phagocytic pathway and in overacidification of the endolysosomal system. Furthermore, ATP8B1 knockdown cells exhibited large multivesicular bodies filled with intraluminal vesicles. Similar phenotypes were observed in CRISPR-generated ATP8B1 knockout cells. Importantly, induction of autophagy led to accumulation of autophagosomes in ATP8B1 knockdown cells. Collectively, our results support a novel role for ATP8B1 in lysosomal fusion in macrophages, a process crucial in the terminal phase of endolysosomal degradation.

Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup.

As neurons age, they show a decrease in their ability to degrade proteins and membranes. Because undegraded material is a source of toxic products, defects in degradation are associated with reduced cell function and survival. However, there are very few dead neurons in the aging brain, suggesting the action of compensatory mechanisms. We show in this work that ageing neurons in culture show large multivesicular bodies (MVBs) filled with intralumenal vesicles (ILVs) and secrete more small extracellular vesicles than younger neurons. We also show that the high number of ILVs is the consequence of the accumulation of cholesterol in MVBs, which in turn is due to decreased levels of the cholesterol extruding protein NPC1. NPC1 down-regulation is the consequence of a combination of upregulation of the NPC1 repressor microRNA 33, and increased degradation, due to Akt-mTOR targeting of NPC1 to the phagosome. Although releasing more exosomes can be beneficial to old neurons, other cells, neighbouring and distant, can be negatively affected by the waste material they contain.

Appearances Can Be Deceiving - Viral-like Inclusions in COVID-19 Negative Renal Biopsies by Electron Microscopy.

Since the discovery of the causative agent for the novel severe acute respiratory syndrome (SARS)–like pneumonia syndrome pandemic that started in China in 2019 (1,2), a coronavirus named SARS coronavirus 2 (SARS-CoV-2), electron microscopy images have populated the medical literature (2) and media outlets alike displaying the characteristic 60–140 nm round particles surrounded by a “corona” of 9–12 nm distinctive spikes (2). Although many of these images were obtained after “ in vitro ” infection of cultured cells with SARS-CoV-2 (2) and are thus likely a true representation of viral particles, we have observed morphologically indistinguishable inclusions within podocytes and tubular epithelial cells both in patients negative for coronavirus disease 2019 (COVID-19) as well as in renal biopsies from the pre–COVID-19 era. Although direct infection of the kidney is theoretically possible, given the presence of angiotensin-converting enzyme 2 (the receptor used by SARS-CoV-2 to gain access to cells) within proximal tubular epithelium (3) and podocytes (4), the virus has not been detected by real-time RT-PCR in urine samples from patients with COVID-19 (5⇓–7). Additionally, for the virus to have access to kidney parenchyma, viremia should occur, and this has only been detected in a minority of patients (6⇓–8). We would, therefore, like to issue a note of caution for inferring viral tissue infection by morphology alone using electron microscopy images from tissues obtained from biopsies or autopsy material in patients with COVID-19. Moreover, caution should be used when interpreting immunohistochemical results, especially within proximal tubules, which are prone to nonspecific staining by a variety of antibodies due to their intense reabsorptive capacity. Additionally, more specific techniques such as immunoelectron microscopy using specific viral antigens (9), or in situ hybridization for viral RNA, are likely necessary to undoubtedly confirm tissue infection in …

Toxin Pores Endocytosed During Plasma Membrane Repair Traffic into the Lumen of MVBs for Degradation

Cells permeabilized by the bacterial pore-forming toxin streptolysin O (SLO) reseal their plasma membrane in a Ca(2+) -dependent manner. Resealing involves Ca(2+) -dependent exocytosis of lysosomes, release of acid sphingomyelinase and rapid formation of endosomes that carry the transmembrane pores into the cell. The intracellular fate of the toxin-carrying endocytic vesicles, however, is still unknown. Here, we show that SLO pores removed from the plasma membrane by endocytosis are sorted into the lumen of lysosomes, where they are degraded. SLO-permeabilized cells contain elevated numbers of total endosomes, which increase gradually in size while transitioning from endosomes with flat clathrin coats to large multivesicular bodies (MVBs). Under conditions that allow endocytosis and plasma membrane repair, SLO is rapidly ubiquitinated and gradually degraded, in a process sensitive to inhibitors of lysosomal hydrolysis but not of proteasomes. The endosomes induced by SLO permeabilization become increasingly acidified and promote SLO degradation under normal conditions, but not in cells silenced for expression of Vps24, an ESCRT-III complex component required for the release of intraluminal vesicles into MVBs. Thus, cells dispose of SLO transmembrane pores by ubiquitination/ESCRT-dependent sorting into the lumen of late endosomes/lysosomes.

Alpha‐ and beta‐synucleins are new diagnostic tools for acute erythroid leukemia and acute megakaryoblastic leukemia

α-Synuclein is a key component of the Lewy body, a large globular protein complex that forms in the nervous system of patients with Parkinson disease and other dementias [1-3]. Since α-synuclein also occurs in megakaryocytic and erythroid lineages [4-7], we wondered what role synucleins had in the hematopoietic system. Therefore, we studied the expression of α-, β-, and γ-synucleins in a comprehensive panel of patient bone marrows and leukemic cell lines. We observed under expression of α-synuclein in the megakaryocytes of myeloproliferative neoplasm (MPN), but not normal reactive marrow (NRM) or myelodysplastic syndrome (MDS). Conversely, we observed over expression of β-synuclein in the blasts of megakaryoblastic leukemias (MegL), but not acute myeloid leukemia (AML) or erythroleukemia (EryL), suggesting that α- and β-synucleins could be useful adjunct markers for the early detection of MDS and the differential diagnosis of EryL and MegL from other AMLs.

Autophagy Contributes to Widespread Neuronal Degeneration in Hamsters Infected with the Echigo-1 Strain of Creutzfeldt-Jakob Disease and Mice Infected with the Fujisaki Strain of Gerstmann-Sträussler-Scheinker (GSS) Syndrome

The authors report here robust autophagy observed by electron microscopy in both the Echigo-1 strain of Creutzfeldt-Jakob disease in hamsters and the Fujisaki strain of Gerstmann-Sträussler-Scheinker disease in mice. In both models, autophagic vacuoles were observed in several cellular compartments. In neuronal cell bodies, autophagic vacuoles of different size were seen. The cytoplasm of some neurons also contained semicircular cisterns equivalent to an early autophasophore. The major target of autophagy was dystrophic neurites, i.e., enlarged neuritic processes—mostly dendrites but also axonal terminals and preterminals. They contained numerous double- or multiple-membrane-bound autophagosomes or autophagolysosomes and large multivesicular bodies. Multivesicular bodies were also observed within autophagic vacuoles, and large multivesicular bodies were seen within synaptic terminals. Some dystrophic neurites was filled almost completely with multivesicular bodies; the latter were occasionally confluent. The authors conclude that autophagy is an important part of neuropathology in prion disease. They also suggest that spongiform vacuoles, a hallmark for the whole group of prion diseases, may in reality originate from autophagic vacuoles.

A minimal hydrodynamic model for a traffic jam in an axon

Traffic jams in long processes of nerve cells (neurons) are linked to such neurodegenerative diseases as Alzheimer's disease and Down syndrome. These traffic jams cause swellings in the neurons' processes that accumulate large amounts of mitochondria, large multi-vesicular bodies, and other types of intracellular organelles, eventually leading to neuron failure and death. The purpose of this paper is to develop a minimal model for a traffic jam in an axon based on a macroscopic (hydrodynamic) approach to modeling of highway traffic and on a macroscopic model of molecular-motor-assisted transport of intracellular organelles on microtubules.

A Raft-derived, Pak1-regulated Entry Participates in α2β1 Integrin-dependent Sorting to Caveosomes

We have previously shown that a human picornavirus echovirus 1 (EV1) is transported to caveosomes during 2 h together with its receptor alpha2beta1 integrin. Here, we show that the majority of early uptake does not occur through caveolae. alpha2beta1 integrin, clustered by antibodies or by EV1 binding, is initially internalized from lipid rafts into tubulovesicular structures. These vesicles accumulate fluid-phase markers but do not initially colocalize with caveolin-1 or internalized simian virus 40 (SV40). Furthermore, the internalized endosomes do not contain glycosylphosphatidylinositol (GPI)-anchored proteins or flotillin 1, suggesting that clustered alpha2beta1 integrin does not enter the GPI-anchored protein enriched endosomal compartment or flotillin pathways, respectively. Endosomes mature further into larger multivesicular bodies between 15 min to 2 h and concomitantly recruit caveolin-1 or SV40 inside. Cell entry is regulated by p21-activated kinase (Pak)1, Rac1, phosphatidylinositol 3-kinase, phospholipase C, and actin but not by dynamin 2 in SAOS-alpha2beta1 cells. An amiloride analog, 5-(N-ethyl-N-isopropanyl) amiloride, blocks infection, causes integrin accumulation in early tubulovesicular structures, and prevents their structural maturation into multivesicular structures. Our results together suggest that alpha2beta1 integrin clustering defines its own entry pathway that is Pak1 dependent but clathrin and caveolin independent and that is able to sort cargo to caveosomes.

B cell receptor signaling governs the subcellular location of Toll-like receptor 9 (86.11)

Abstract Toll-like receptor 9 (TLR9), an innate immune system receptor for hypomethylated CpG DNA motifs, has gained substantial attention recently due to its unique ability to promote the activation of autoreactive B cells in response to DNA-containing antigens. Many of the autoantigens targeted in systemic lupus erythematosus (SLE) contain DNA. Moreover, TLR9 has been shown to play a critical role in regulating anti-DNA autoantibody production in mouse models of lupus by mechanisms that involve the simultaneous engagement of TLR9 and the B cell receptor (BCR). However, the cellular and molecular basis of the interaction between TLR9 and the BCR is not known and given the spatial segregation of the BCR on the plasma membrane and TLR9 in early endosomal compartments, it is not obvious how synergy is achieved. Here we show that when activated independently the BCR and TLR9 signal from discrete subcellular compartments, the BCR initially from the plasma membrane and later from large multivesicular bodies (MVB) and TLR9 from multiple endosomes. Remarkably, stimulation of B cells simultaneously through the BCR and TLR9 triggers the recruitment of the TLR9-containing endosomes to the MVB by a Src-family kinase-dependent, MyD88-independent mechanism resulting in a synergistic response. Thus, the BCR triggers the recruitment of TLR9- containing vesicles to the MVB presumably to allow TLR9 to survey the BCR bound antigen for its DNA ligand, which if present, results in TLR9 activation and enhanced B cell responses. Our study adds a new spatial dimension to our view of TLR9 signaling in the B cells, potentially providing a new arena for therapeutic drug design.

Regulatory role of kinases and phosphatases on the internalisation of caveolae in HepG2 cells

The caveolar cycle is thought to be regulated by synchronised function of kinases and phosphatases. Using ocadaic acid – a serine/threonine protein phosphatase inhibitor – and an inhibitor of tyrosine phosphatase (sodium orthovanadate) we have followed the internalisation of caveolae. Since albumin binding to its receptor (gp60) can induce pinching off of caveolae from the plasma membrane, we also used this physiological ligand to induce the internalisation. Our confocal microscopic results show that both ocadaic acid and vanadate treatments have significantly decreased caveolin (caveolin-1 and -2) labelling on the cell surface, while the cytoplasmic labelling became much stronger. Quite often large, strongly labelled “granules” appear at the perinuclear region. Very strong caveolin labelling was detected along the actin–cytoskeleton suggesting that caveolae might move along these filaments. Our electron microscopic results also show an intensive caveolae pinching off from the plasma membrane. After ocadaic acid and vanadate treatments the number of surface connected vesicles (caveolae) decreases. At the same time, large multivesicular bodies (termed caveosomes) appear in the perinuclear area of the cytoplasm. By immunoprecipitation and Western blot analysis we detect an increased tyrosine phosphorylation of a ∼29 kDa protein in ocadaic acid and vanadate treated samples. This protein was identified as caveolin-2. No significant change in the tyrosine phosphorylation of caveolin-1 was found. From these data we can conclude that caveolae internalisation is regulated by phosphorylation of caveolin-2.

Ultrastructural changes in the corpus allatum after azadirachtin and 20-hydroxyecdysone treatment in adult females of Labidura riparia (Dermaptera)

In previous reports, we have shown that the injection of azadirachtin (AZA) as well as 20-hydroxyecdysone (20E) into vitellogenic females of Labidura riparia induces inhibition of vitellogenin synthesis and ovarian development. Juvenile hormone (JH) treatment rescues vitellogenin synthesis and ovarian growth (Sayah et al., 1995, 1996). In this work, we have studied ultrastructural changes of corpus allatum (CA) after injection of 200, 400, and 600 ng of 20E or 1, 3, and 5 μg of AZA. CA cells exhibit signs of inactivity in both AZA and females treated with 20E at doses of 3 μg and 400 ng, respectively. Conspicuous cytological effects consisting of multivesicular bodies with dense contents, abnormally large intercellular spaces comprising myelinic structures, and rare smooth endoplasmic reticula occured in cytoplasm of CA glandular cells in both experimental females. However, the CA ultrastructure of females injected with 20E differs from CA cells of females injected with AZA in having a cytoplasm containing numerous electron-lucent intracellular areas and marked glycogen zones. They also differ in having abundant microtubules and well-developed junctional membranes. At a dose of 600 ng of 20E or 5 μg of AZA, the intensity of the cytotoxic effects is more apparent. CA cells display pycnotic nuclei, spherical mitochondria, large multivesicular bodies, and vacuolization of the cytoplasm. These results are discussed and compared with observations made on other insect species.

Ultrastructure of the surface structures and haptor of Empleurosoma pyriforme (Ancyrocephalinae; Monopisthocotylea: Monogenea) from the gills of the teleost fish Therapon jarbua.

Infections with Empleurosoma pyriforme occur between successive secondary gill lamellae on both sides of the primary lamella of Therapon jarbua. The haptoral disc bears two pairs of anchors and a pair of connecting transverse bars. The attachment of the parasite to the host gill causes inflammation, erosion and degeneration of the gill epithelia. The ventral anchors consist of an inner core of irregularly arranged, electron-dense fibrils and a smooth outer core of electron-lucent fibrils, whereas the surface of the dorsal anchors is ridged. Both the dorsal and the ventral anchors may be extended or withdrawn. The connecting transverse bars consist of longitudinally arranged fibrils in an electron-dense matrix, whereas the tendons consist of fibrils, supported in a less electron-dense matrix, which interconnect the anchor erector-protractor muscles and the haptor muscles. Two types of perikarya are present. The less common type contain large multivesicular bodies and small electron-dense granules and are located only in the haptor region. The second and more common perikarya are present throughout the body surface. The cytoplasmic syncytium contains numerous electron-dense granules and electron-lucent vesicles. Beneath the syncytium, unicellular epidermal gland cells contain electron-dense granules. Neurones containing numerous electron-dense vesicles are present in the haptor region. Uniciliate presumed sensory receptors are distributed over the body surface. Groups of ciliated sensory structures are present in the forebody. Ciliated and non-ciliated presumed sensory receptors are present in the sleeve cavity of the anchors, on the haptor and in the vicinity of the oral apertures.

RdgE: a novel retinal degeneration mutation in Drosophila melanogaster.

We report isolating the Drosophila retinal degeneration E (rdgE) mutation. The hypomorphic rdgE1 allele causes rapid photoreceptor degeneration in light and a slower rate of degeneration when the flies are raised in constant darkness. The rdgE1 flies exhibited an electrophysiological light response that decreased with age, coinciding with the degeneration. This suggests that degeneration caused the loss of the light response. We determined that the ninaE (rhodopsin) mutation, but not norpA [phospholipase C (PLC)], slowed the rdgE-dependent degeneration. This was consistent with the light-enhanced degeneration, but revealed that the degeneration is independent of the PLC-mediated phototransduction cascade. Transmission electron microscopy revealed that rdgE1 photoreceptors exhibited a number of vesicular transport defects including unpacking/vesiculation of rhabdomeres, endocytosis of novel vesicles by photoreceptors, a buildup of very large multivesicular bodies, and an increased amount of rough endoplasmic reticulum. We determined that the rdgE null phenotype is a late embryonic lethality. Therefore, rdgE+ is required in cells outside of the retina, quite possibly in a large number of neurons. Thus, rdgE may define a mutational class that exhibits both light-enhanced retinal degeneration and a recessive null lethality by perturbing neuronal membrane biosynthesis and/or recycling.

Ultrastructure of the surface structures and electron immunogold labeling of peptide immunoreactivity in the nervous system of Pseudothoracocotyla indica (Polyopisthocotylea: Monogenea).

Transmission electron microscope studies of the tegument of the tropical marine fish monogenean parasite Pseudothoracocotyla indica describe surface specialisations and detail the ultrastructure of the tegument and the haptor. The tegument consists of a syncytium, numerous electron-dense granules, electron-lucent vesicles and large multivesicular bodies. The posterior tegumental syncytium is infolded to form tegumental ridges that are present on both the ventral and dorsal surfaces. A thin coat of glycocalyx is present on the tegument surface. In contrast, the tegumental syncytium of the haptor is relatively thin, containing electron-dense granules and various-sized electron-lucent vesicles. Exocytosis of the electron-dense and electron-lucent vesicles apparently occurs in the syncytium of the haptor and general body surface. Tegumental damage was observed on the dorsal surface in the mid-body region and may possibly have been due to natural mechanical forces. The haptor consists of electron-dense clamp sclerites embedded within a matrix covered by the tegumental syncytium. The sclerites are connected to each other and to the basal lamina by radially oriented muscle fibres. The haptor is richly supplied with non-myelinated nerve axons. Both uniciliated and non-ciliated presumed sensory structures are present on the body surface and haptor. Uniciliated sensory structures were found mainly around the oral sucker. Groups of neurons and nerve processes containing neurosecretory vesicles were frequently observed in the vicinity of the clamps. Electron immunogold labelling studies demonstrated that neuropeptide F [NPF (Moniezia expansa)] immunoreactivity was confined to electron-dense-cored neurosecretory vesicles in nerve fibres from the posterior and haptor regions of the fluke.

Alterations induced by the antifungal compounds ketoconazole and terbinafine in Leishmania.

The antiproliferative effects and ultrastructural alterations induced in vitro by two antifungal compounds, the azole ketoconazole and the allylamine terbinafine on Leishmania amazonensis are reported. Promastigotes treatment with ketoconazole and terbinafine induced growth arrest and cell lysis in 72 hours. Combination of the two agents produced additive effects on promastigote axenic growth and synergistic effects on intracellular amastigote proliferation. The amastigotes, either axenically grown or infecting murine macrophages, were about 100-fold more sensitive to the drugs. These compounds induced the appearance of large multivesicular bodies, especially after ketoconazole treatment, increased amount of lipid inclusions as well as numerous, polymorphic volutin granules, particularly in terbinafine-treated cells. Multivesicular bodies were observed in close apposition with organelles such as mitochondria, which also showed alterations in the distribution and appearance of cristae, and the formation of paracrystalline arrays within the matrix. Some cells presented large portions of cytoplasm wrapped by endoplasmic reticulum and many parasites also presented myelin-like endoplasmic reticulum profiles. Such alterations together with the strong acid phosphatase activity observed in the multivesicular bodies and volutin granules may indicate the existence of an unusual autophagic process in cells treated with ergosterol biosynthesis inhibitors.

Changes in the nucleus, endoplasmic reticulum, Golgi apparatus, and acrosome during spermiogenesis in the waterstrider, Gerris najas deg. (Heteroptera : Gerridae)

During spermiogenesis in Gerris najas (Heteroptera : Gerridae), wide tubules of endoplasmic reticulum are closely associated with the trans-tubular network of the Golgi apparatus. This relationship, formerly observed in spermatocytes of this species, exists also in spermatids almost from the beginning to very late stages. Different functions of the Golgi apparatus can be distinguished. At the time the acrosomal vesicle is formed, unusually large multivesicular bodies originate within the Golgi apparatus. Morphogenetic transformation of the acrosome from a huge sphere, measuring about 6.5 μm in diameter, to a long ;2.5 mm) needle-like structure, is a very complicated process, during which cytoplasmiz! components become incorporated into the definitive acrosome.

Vasopressin- and cAMP-induced changes in ultrastructure of isolated perfused inner medullary collecting ducts

Studies were performed to correlate arginine vasopressin (AVP)-induced changes in epithelial ultrastructure with changes in osmotic water permeability in isolated perfused rat terminal inner medullary collecting ducts (tIMCD). The tubules were perfused in three time periods, i.e., a 40-min basal period, a 40-min period with 0.1 nM AVP in the bath, and a 60-min withdrawal period. In each phase, the osmotic water permeability (Pf) was measured, and the perfused tubules were fixed for electron microscopy. AVP caused a four- to eightfold increase in Pf and induced several ultrastructural changes as follows: increased cell height of IMCD cells, expansion of the intercellular spaces, formation of large vacuoles, and increased coated pit density in the apical plasma membrane [from 0.6 +/- 0.2 (n = 6) to 2.9 +/- 0.3 (n = 7) pits/100 microns membrane length]. During AVP withdrawal, Pf decreased toward the basal value in association with partial reversal of the ultrastructural changes including a decrease in coated pit density to 1.0 +/- 0.2 (n = 4). Stimulation with 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) (0.1 mM) produced similar changes in Pf. Coated pit density increased to 2.1 +/- 0.4 (n = 4) after cAMP stimulation and after cAMP withdrawal decreased to 1.2 +/- 0.2 (n = 6). In contrast to stimulation with AVP, cAMP stimulation did not result in dilated intercellular spaces or formation of large vacuoles. The only ultrastructural feature that directly correlated with the water permeability was the density of coated pits in the apical membrane. Organelles involved in the endocytic pathway were studied with cationized ferritin or albumin-gold in the luminal perfusate. At the end of 40 min basal perfusion or AVP stimulation, luminal tracer was found almost exclusively in large multivesicular bodies (MVB). Tubules perfused with tracer during AVP withdrawal demonstrated rapid tracer accumulation in small vesicles and small MVB within 3-5 min, a time point corresponding to the rapid phase of Pf decrease. Later (30-60 min) the label was mainly confined to large MVB. Occasionally during AVP stimulation or withdrawal, small coated vesicles and smooth vesicles with coated extensions were noted to contain tracer. The data demonstrate AVP-mediated coated pit formation and cellular changes and show very rapid internalization of apical membrane after AVP withdrawal.

The ultrastructure of the tegument and clamp attachment organ of Gotocotyla bivaginalis (Monogenea, polyopisthocotylea)

The tegument of Gotocotyla bivaginalis consists of a syncytium, perikarya and four kinds of secretory bodies, viz. electron-dense granules, moderately electron-dense vesicles, electronlucent vesicles and large multivesicular bodies occurring in the ratio of 83:50:40:1. Microvillus-like structures, finger-like projections and a thin coat of glycocalyx occur on the tegument. In contrast, the syncytium of the clamp is relatively thin and irregularly folded, containing only three kinds of secretory bodies, viz. electron-dense granules, a few moderately electron-dense granules and several large electronlucent vacuole-like structures in the ratio of 15:2:5. Exocytosis of the electron-dense and -lucent vesicles is apparently prevalent both in the syncytium of the haptor and general body surface. Tegumental damages induced by natural mechanical forces appear to occur in some regions of the syncytium of the clamps/ haptor. Sclerites of the clamp are electron-dense and they are interconnected by the basal lamina, tendon and radial muscle fibres. Interstitial space is absent between the sarcolemma of adjacent muscle fibres of the clamp. Groups of neurons and non-myelinated nervous processes can be seen in the vicinity of the clamps. The axon is subdivided by invaginations of the neurolemma. Presumed non-ciliated mechano-receptors occur on the body surface.

Autocrine motility factor and its receptor: role in cell locomotion and metastasis.

The ability to locomote and migrate is fundamental to the acquisition of invasive and metastatic properties by tumor cells. Autocrine motility factor (AMF) is a 55 kD cytokine produced by various tumor cells which stimulates their in vitro motility and in vivo lung colonizing ability. AMF stimulates cell motility via a receptor-mediated signalling pathway. Signal transduction following binding of AMF to its receptor, a cell surface glycoprotein of 78 kD (gp78) homologous to p53, is mediated by a pertussis toxin sensitive G protein, inositol phosphate production and the phosphorylation of gp78. Cell surface gp78 is localized to the leading and trailing edges of motile cells but following cell permeabilization is found within an extended network of intracellular tubulovesicles. Gp78 tubulovesicles colocalize with microtubules and extension of the tubulovesicular network to the cell periphery is dependent on the presence of intact microtubules. Gp78 labeled vesicles can be induced to translocate between the cell center and periphery by altering intracellular pH as previously described for tubulovesicles labeled by fluid phase uptake. Anti-gp78 mAb added to viable motile cells is localized to large multivesicular bodies which, with time, relocate to the leading edge. Binding of AMF to its receptor induces signal transduction, similar to chemotactic stimulation of neutrophil mobility, as well as the internalization and transport of its receptor to the leading edge stimulating pseudopodial protrusion and cell motility.