Accumulation Of Vesicles Research Articles

The Spectral-Domain Optical Coherence Tomography Findings Associated with the Morphological and Electrophysiological Changes in a Rat Model of Retinal Degeneration, Rhodopsin S334ter-4 Rats.

Purpose To characterize the spectral-domain optical coherence tomography (SD-OCT) findings of the rhodopsin S334ter transgenic rats (line 4) in relation to the morphologic and electroretinographic features. Materials and Methods Rhodopsin S334ter transgenic rats (line 4) were employed as a model of retinal degeneration. The Sprague-Dawley (SD) rats were used as a wild-type control. SD-OCT (Micron IV®; Phoenix Research Labs, Pleasanton, CA, USA) was performed on the S334ter rats (line 4) from postnatal days (P) 13−110. The longitudinal changes of the SD-OCT images were analyzed both qualitatively and quantitatively in comparison to those of SD rats. The SD-OCT images were also compared to the histological and electron microscopic findings from examination performed on P 22, 36, and 61. Full field combined rod and cone electroretinography (ERG) was performed and the relationship between the thickness of the retinal sublayers and the amplitudes of the a- and b-waves was further analyzed. Results The photoreceptor inner and outer segment layer became diffusely hyperreflective in the SD-OCT images of the S334ter rats; these findings were not observed in the SD rats. This hyperreflective change corresponded to the degenerated inner and outer segments and the accumulation of the extracellular vesicles in the interphotoreceptor matrix. Quantitatively, the retinal outer sublayer and the photoreceptor sublayer in the S334ter rats became progressively thinner in comparison to those in the SD rats; the difference was statistically significant. The amplitudes of both the a- and b-waves on ERG were severely deteriorated in the S334ter rats. Discussion The SD-OCT images in the S334ter rats noninvasively provided information regarding the pathological changes in the photoreceptors and the longitudinal changes of both qualitative and quantitative changes during retinal degeneration in the S334ter rats (line 4). The pathological features of the photoreceptor inner and outer segments can be detected on SD-OCT as diffuse hyperreflective changes in the photoreceptor layer.

Phaseolin ingestion affects vesicular traffic causing oxidative stress in the midgut of Callosobruchus maculatus larvae.

It has been reported that phaseolin, the major storage globulin of the common bean (Phaseolus vulgaris), is toxic to Callosobruchus maculatus larvae, an Old World bruchid beetle that is not capable of infesting this New World edible bean. It has also been demonstrated that vicilin, the major storage globulin found in cowpea (Vigna unguiculata) seeds, is absorbed through receptor-mediated endocytosis in the insect midgut. A putative vicilin receptor has been purified and showed high homology to α-tocopherol transfer protein. However, the ingestion of a variant vicilin purified from C. maculatus resistant seeds inhibits transcytosis, resulting in the accumulation of vicilins in the midgut cells and ultimately antibiosis. In the present work, we studied the cellular up-take of phaseolin in C. maculatus larvae with the aim of discovering if this protein is also capable of inhibiting endocytic traffic in the enterocytes. FITC-labelled vicilin and FITC-labelled phaseolin were incorporated into the diet of the larvae at a physiological concentration of 0.5% w/w. The fate of labelled and non-labelled globulins was monitored by confocal microscopy. Here we demonstrated that phaseolin is also endocytosed by enterocytes causing an accumulation of endocytic vesicles in the midgut when compared to the ingestion of vicilin obtained from a susceptible V. unguiculata cultivar. From the results obtained for HNE, MDA and TBARS, a pro-oxidative scenario was established in the intestinal epithelial cells of the larvae, which may explain the deleterious effect observed in larvae developing inside P. vulgaris seeds.

Arabidopsis class I formins control membrane-originated actin polymerization at pollen tube tips.

A population of dynamic apical actin filaments is required for rapid polarized pollen tube growth. However, the cellular mechanisms driving their assembly remain incompletely understood. It was postulated that formin is a major player in nucleating apical actin assembly, but direct genetic and cytological evidence remains to be firmly established. Here we found that both Arabidopsis formin 3 (AtFH3) and formin 5 (AtFH5) are involved in the regulation of apical actin polymerization and actin array construction in pollen tubes, with AtFH3 playing a more dominant role. We found that both formins have plasma membrane (PM) localization signals but exhibit distinct PM localization patterns in the pollen tube, and loss of their function reduces the amount of apical actin filaments. Live-cell imaging revealed that the reduction in filamentous actin is very likely due to the decrease in filament elongation. Furthermore, we found that the rate of tip-directed vesicle transport is reduced and the pattern of apical vesicle accumulation is altered in formin loss-of-function mutant pollen tubes, which explains to some extent the reduction in pollen tube elongation. Thus, we provide direct genetic and cytological evidence showing that formin is an important player in nucleating actin assembly from the PM at pollen tube tips.

Tetrahymena promotes interactive transfer of carbapenemase gene encoded in plasmid between fecal Escherichia coli and environmental Aeromonas caviae.

Tetrahymena can facilitate plasmid transfer among Escherichia coli or from E. coli to Salmonella Enteritidis via vesicle accumulation. In this study, whether ciliates promote the interactive transfer of plasmids encoding blaIMP-1 between fecal E. coli and environmental Aeromonas caviae was investigated. Both bacteria were mixed with or without ciliates and incubated overnight at 30°C. The frequency of plasmid-acquired bacteria was estimated by colony counts using an agar plate containing ceftazidim (CAZ) followed by determination of the minimum inhibitory concentration (MIC). Cultures containing ciliates interactively transferred the plasmid between E. coli and Aeromonas with a frequency of 10-4 to 10-5 . All plasmid-acquired bacteria showed a MIC against CAZ of >128 μg/mL and the plasmid transfer was confirmed by PCR amplification of the blaIMP-1 gene. Fluorescent observation showed that both bacteria accumulated in the same vesicle and that transwell sequestering significantly decreased the transfer frequency. Although ciliates preferentially ingested E. coli rather than A. caviae, both bacteria were co-localized into the same vesicles of ciliates, indicating that their meeting is associated with the gene transfer. Thus, ciliates interactively promote plasmid transfer between E. coli and A. caviae. The results of this study will facilitate control of the spread of multiple-antibiotic resistant bacteria.

CD2-Associated Protein Contributes to Hepatitis C, Virus Propagation and Steatosis by Disrupting Insulin Signaling.

Chronic hepatitis C virus (HCV) infection can result in steatosis, a condition displaying aberrant accumulation of neutral lipid vesicles, the component of lipid droplets (LDs), which are essential for HCV assembly. However, the interplay between HCV infection and steatosis remains unclear. Here, we show that HCV‐infected cells have higher levels of CD2‐associated protein (CD2AP), which plays two distinct, yet tightly linked, roles in HCV pathogenesis: Elevated CD2AP binds to nonstructural protein 5A (NS5A) and participates in the transport of NS5A to LDs to facilitate viral assembly; Up‐regulated CD2AP also interacts with casitas B‐lineage lymphoma (b) (Cbl/Cbl‐b) E3 ligases to degrade insulin receptor substrate 1 (IRS1), which, in turn, disrupts insulin signaling and increases LD accumulation through the IRS1/protein kinase B (Akt)/adenosine monophosphate‐activated protein kinase (AMPK)/hormone‐sensitive lipase (HSL) signaling axis to accommodate viral assembly. In the HCV‐infected mouse model, CD2AP expression is up‐regulated during the chronic infection stage and this up‐regulation correlates well with liver steatosis. Importantly, CD2AP up‐regulation was also detected in HCV‐infected human liver biopsies showing steatosis compared to non‐HCV‐infected controls. Conclusion: CD2AP is indicated as a protein up‐regulated by HCV infection, which, in turn, stimulates HCV propagation and steatosis by disrupting insulin signaling; targeting CD2AP may offer an opportunity for alleviating HCV infection and its associated liver pathology. (Hepatology 2018;XX:XXX‐XXX.)

Nanoparticle-Lipid Interaction: Job Scattering Plots to Differentiate Vesicle Aggregation from Supported Lipid Bilayer Formation

The impact of nanomaterials on lung fluids, or on the plasma membrane of living cells, has prompted researchers to examine the interactions between nanoparticles and lipid vesicles. Recent studies have shown that nanoparticle-lipid interaction leads to a broad range of structures including supported lipid bilayers (SLB), particles adsorbed at the surface or internalized inside vesicles, and mixed aggregates. Currently, there is a need to have simple protocols that can readily evaluate the structures made from particles and vesicles. Here we apply the method of continuous variation for measuring Job scattering plots and provide analytical expressions for the scattering intensity in various scenarios. The result that emerges from the comparison between experiments and modeling is that electrostatics play a key role in the association, but it is not sufficient to induce the formation of supported lipid bilayers.

Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells.

The ATP-binding cassette transporter G1 (ABCG1) is a cholesterol lipid efflux pump whose role in tumor growth has been largely unknown. Our transcriptomics revealed that ABCG1 was powerfully expressed in rapidly metastatic, aggregative colon cancer cells, in all the ABC transporter family members. Coincidently, genetic amplification of ABCG1 is found in 10–35% of clinical samples of metastatic cancer cases. Expression of ABCG1 was further elevated in three-dimensional tumoroids (tumor organoids) within stemness-enhancing tumor milieu, whereas depletion of ABCG1 lowered cellular aggregation and tumoroid growth in vitro as well as hypoxia-inducible factor 1α in cancer cells around the central necrotic areas in tumors in vivo. Notably, depletion of ABCG1 triggered the intracellular accumulation of extracellular vesicles (EVs) and regression of tumoroids. Collectively, these data suggest that ABCG1 plays a crucial role in tumorigenesis in metastatic cancer and that depletion of ABCG1 triggers tumor regression with the accumulation of EVs and their derivatives and cargos, implicating a novel ABCG1-targeting therapeutic strategy by which redundant and toxic substances may be accumulated in tumors leading to their regression.

Effects of mono- and di-valent metal cations on the morphology of lipid vesicles

Lipid vesicles are an attractive model membrane experimental platform that is widely used in a biological context. The stability of vesicles can affect their performance and depends on various experimental conditions. How bio-related ions affect vesicle morphology is poorly understood in some cases. Herein, we investigated changes in vesicle morphology influenced by cation in the static and flowing environments. The effects of different mono- and di-valent metal cations on the morphology of lipid vesicles were systematically studied using the various techniques. The results showed that divalent cations caused significant aggregation or fusion of lipid vesicles, but monovalent cations had little effect on the vesicle morphology. Cation binding increased the net surface potential of vesicles, leading to changes in the zeta potential. The same qualitative kinetics were observed for cations that had the same valence at the same ionic strength. However, different types of cations gave different quantitative effects. The order of the ability to destroy the vesicle morphology was Cu2+ > Mg2+ > Ca2+ > Na+ > K+. These results are of practical value in the use of lipid vesicles as a bionic model, and help to shed light on the role of ions at membrane surfaces and interfaces.

A Comparative Study of Phosphatidylcholine versus Phosphatidylserine-Based Solid Supported Membranes for the Preparation of Liposome-Rich Interfaces.

Solid supported membranes (SSMs) are usually formed by an hybrid octadecanethiol/phosphatidylcholine (PC) bilayer supported by a gold electrode. Recently, it was shown that phosphatidylserine (PS) in place of PC can promote a more effective accumulation of lipid vesicles on the SSM surface when Ca2+ and Mg2+ ions are present in the external environment. Here we performed a detailed comparative study of the vesicle adsorption process onto PC- and PS-SSMs by employing surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). SPR analysis has demonstrated a higher affinity of the PS-SSM surface for the phospholipid vesicles. Both SPR and EIS measurements suggest that adsorption of lipid vesicles on the PC-SSM tends to a saturating value, whereas a continuous and progressive vesicle adsorption occurs on the PS-SSM surface following subsequent liposome additions. AFM analysis pointed out a systematic flattening of the adsorbed vesicles on the PS-SSM surface. We interpreted our results as due to the strong coordinating action of the high amount of divalent cations accumulated at the negatively charged PS-SSM surface, whereas a lower amount of cations is present on the dipolar PC-SSM surface, which can therefore adsorb only a limited number of vesicles.

Rab‐H1b is essential for trafficking of cellulose synthase and for hypocotyl growth in Arabidopsis thaliana

Cell-wall deposition of cellulose microfibrils is essential for plant growth and development. In plant cells, cellulose synthesis is accomplished by cellulose synthase complexes located in the plasma membrane. Trafficking of the complex between endomembrane compartments and the plasma membrane is vital for cellulose biosynthesis; however, the mechanism for this process is not well understood. We here report that, in Arabidopsis thaliana, Rab-H1b, a Golgi-localized small GTPase, participates in the trafficking of CELLULOSE SYNTHASE 6 (CESA6) to the plasma membrane. Loss of Rab-H1b function resulted in altered distribution and motility of CESA6 in the plasma membrane and reduced cellulose content. Seedlings with this defect exhibited short, fragile etiolated hypocotyls. Exocytosis of CESA6 was impaired in rab-h1b cells, and endocytosis in mutant cells was significantly reduced as well. We further observed accumulation of vesicles around an abnormal Golgi apparatus having an increased number of cisternae in rab-h1b cells, suggesting a defect in cisternal homeostasis caused by Rab-H1b loss function. Our findings link Rab GTPases to cellulose biosynthesis, during hypocotyl growth, and suggest Rab-H1b is crucial for modulating the trafficking of cellulose synthase complexes between endomembrane compartments and the plasma membrane and for maintaining Golgi organization and morphology.

Rab6 promotes insulin receptor and cathepsin trafficking to regulate autophagy induction and activity in Drosophila.

The self-degradative process of autophagy is important for energy homeostasis and cytoplasmic renewal. This lysosome-mediated pathway is negatively regulated by the target of rapamycin kinase (TOR) under basal conditions, and requires the vesicle trafficking machinery regulated by Rab GTPases. However, the interactions between autophagy, TOR and Rab proteins remain incompletely understood in vivo Here, we identify Rab6 as a critical regulator of the balance between TOR signaling and autolysosome function. Loss of Rab6 causes an accumulation of enlarged autophagic vesicles resulting in part from a failure to deliver lysosomal hydrolases, rendering autolysosomes with a reduced degradative capacity and impaired turnover. Additionally, Rab6-deficient cells are reduced in size and display defective insulin-TOR signaling as a result of mis-sorting and internalization of the insulin receptor. Our findings suggest that Rab6 acts to maintain the reciprocal regulation between autophagy and TOR activity during distinct nutrient states, thereby balancing autophagosome production and turnover to avoid autophagic stress.

Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation.

Macroautophagy is an intracellular degradation process that requires multiple autophagy-related (ATG) genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified TMEM41B as a novel ATG gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER). It has a conserved domain also found in vacuole membrane protein 1 (VMP1), another ER multispanning membrane protein essential for autophagy, yeast Tvp38, and the bacterial DedA family of putative half-transporters. Deletion of TMEM41B blocked the formation of autophagosomes at an early step, causing accumulation of ATG proteins and small vesicles but not elongating autophagosome-like structures. Furthermore, lipid droplets accumulated in TMEM41B-knockout (KO) cells. The phenotype of TMEM41B-KO cells resembled those of VMP1-KO cells. Indeed, TMEM41B and VMP1 formed a complex in vivo and in vitro, and overexpression of VMP1 restored autophagic flux in TMEM41B-KO cells. These results suggest that TMEM41B and VMP1 function together at an early step of autophagosome formation.

LRRK2 deficiency impairs trans-Golgi to lysosome trafficking and endocytic cargo degradation in human renal proximal tubule epithelial cells.

Defects in vesicular trafficking underlie a wide variety of human diseases. Genetic disruption of leucine-rich repeat kinase 2 (LRRK2) in rodents results in epithelial vesicular trafficking errors that can also be induced by treatment of animals with LRRK2 kinase inhibitors. Here we demonstrate that defects in human renal cells lacking LRRK2 phenocopy those seen in the kidneys of Lrrk2 knockout mice, characterized by accumulation of intracellular waste vesicles and fragmentation of the Golgi apparatus. This phenotype can be recapitulated by knockdown of N-ethylmaleimide-sensitive factor, which physically associates with LRRK2 in renal cells. Deficiency in either protein leads to a defect in trans-Golgi to lysosome protein trafficking, which compromises the capacity of lysosomes to degrade endocytic and autophagic cargo. In contrast, neither bulk endocytosis nor autophagic flux are impaired when LRRK2 is acutely knocked down in normal immortalized human kidney (HK2) cells. These data collectively suggest that the primary renal defect caused by LRRK2 deficiency is in protein trafficking between the Golgi apparatus and late endosome/lysosome, which leads to progressive impairments in lysosomal function.

Photo-Controlled Polymerization-Induced Self-Assembly (Photo-PISA): A Novel Strategy Using In Situ Bromine-Iodine Transformation Living Radical Polymerization.

A series of hydrophilic poly(poly(ethylene glycol) methyl ether methacrylate) (PPEGMA) macroinitiators and stabilizers are synthesized in methanol through in situ photo-controlled bromine-iodine transformation living radical polymerization, where ethyl α-bromophenylacetate (EBPA) is the initial initiator and is converted to an iodo-type initiator in the presence of NaI. The subsequent photo-controlled polymerization-induced self-assembly (photo-PISA) process is achieved by adding a second monomer, hydrophobic benzyl methacrylate (BnMA), under irradiation with blue light emitting diode (LED) light at room temperature. The effect of the target degree of polymerization (DP) of PPEGMA, PBnMA, as well as the solids content on the self-assembly behavior of block copolymer PPEGMA-b-PBnMA is evaluated by gel permeation chromatography (GPC), nuclear magnetic resonance (NMR) spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS) characterization. Resulting uniform spherical micelles and vesicle aggregates are observed.

In vitro leishmanicidal effects of the anti-fungal drug natamycin are mediated through disruption of calcium homeostasis and mitochondrial dysfunction.

Natamycin, a Food and Drug Administration approved anti-fungal drug, and also used as a food additive was evaluated for anti-leishmanial activity since it is known to specifically bind to ergosterol, which is essential to these parasites but absent in mammals. Promising anti-proliferative activity was observed in both promastigote and amastigote forms of the parasite with IC50 values of 15 and 8µM respectively and a selective index of 12.5. The ultrastructural effects of natamycin on both forms of the parasite and physiological effects on promastigotes were studied in detail for the first time. Electron microscopic observations in treated cells revealed sub-cellular changes like plasma membrane alterations, accumulation of vesicles in the flagellar pocket and extensive mitochondrial damage. Natamycin treatment in promastigotes resulted in elevation of cytosolic calcium (Ca2+) levels which caused irreversible loss of mitochondrial membrane potential. This resulted in depletion of cellular ATP levels along with ROS generation finally leading to apoptosis-like and necrotic cell death. In view of our observations along with the safety profile of an existing anti-fungal drug, natamycin may be further investigated for repurposing it as a promising drug candidate against Leishmaniasis.

Abstract 43: Regulation of autophagy in bone metastatic breast cancer cells

Abstract Bone metastasis of breast cancer is a significant cause of patient mortality. Recent studies suggest that metastatic cancer cells induce autophagy to survive metabolic stress. During autophagy, cytoplasmic components and damaged organelles are captured by autophagosomes followed by lysosomal fusion and degradation, releasing metabolites as energy sources to meet metabolic demands. Although the components of the autophagy pathway have been well characterized, the regulatory mechanisms of autophagy in metastatic cancer cells remain unknown. Previously, we have shown that Runt-related transcription factor-2 (Runx2) promotes cell survival, bone metastasis, and osteolysis. Using a bone metastatic isogenic variant of breast cancer MDA-MB-231, we examined levels of the autophagosome specific marker LC3B to define the regulation of autophagy during bone metastasis. Additionally, we examined whether Runx2 regulates autophagy for increased cell survival in the bone microenvironment. Microscopic and biochemical studies showed elevated levels of autophagic flux among bone derived cells relative to parental breast cancer cells. Interestingly, we also observed that Runx2 enhanced the turnover of autophagic vesicles while Runx2 silencing resulted in accumulation of vesicles due to reduced turnover. Interestingly, Runx2 knockdown or inhibition of autophagy in bone derived cells increased AMPK levels suggesting higher levels of cellular stress as a consequence of impaired autophagy. In addition to AMPK activity, MAP kinase mutations have been demonstrated to result in constitutive activation of the autophagy pathway. Treatment with the MEK inhibitor PD184161 resulted in accumulation of LC3B-II in control cells. Furthermore, Runx2 knockdown in bone derived cells display lower levels of ERK activity relative to controls. These results suggest that Runx2/ERK signaling is critical for autophagy in metastatic breast cancer cells. Our mechanistic studies revealed that Runx2 promotes autophagy by increasing acetylation of α-tubulin sub-units of microtubules and enhancing trafficking of autophagic vesicles. Introduction of a mutant α-tubulin construct incapable of being acetylated resulted in the accumulation of autophagic vesicles in control cells, similar to silencing of Runx2. Inhibition of autophagy resulted in decreased adhesion, migration, and survival of Runx2 knockdown cells. Furthermore, analysis of LC3B protein in clinical breast cancer specimens and tumor xenografts revealed significant association between high Runx2 and low LC3B protein levels. Our studies reveal a novel regulatory mechanism of autophagy via Runx2 and provide insights into the role of autophagy in bone metastatic breast cancer cells. Citation Format: Ahmad H. Othman, Manish Tandon, Vivek Ashok, Marcus Winogradzki, Gary Stein, Jitesh Pratap. Regulation of autophagy in bone metastatic breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 43.

ALA inhibits pear pollen tube growth through regulation of vesicle trafficking

Abstract 5-Aminolevunic acid, a natural plant growth regulator, can thin pear fruits by inhibiting pollen tube growth through Ca2+-ATPase-mediated Ca2+ efflux. Here, using a non-invasive micromeasurement technology, we detected the occurrence of an ALA-induced Ca2+ efflux. Transmission electron microscope observations showed that a disruption of the intracellular Ca pool, including endoplasmic reticulum and mitochondria, was also linked to ALA-decreased Ca2+ in pollen tubes. Then we further explored the mechanisms how ALA-decreased Ca2+ inhibits pollen tube growth. We observed that ALA-inhibited pollen tube growth was accompanied by tube swelling and disruption in an ALA concentration-dependent manner. Ultrastructural studies further showed that ALA increased the number of vesicles, and the vesicle aggregation resulted in the morphological abnormality of pollen tubes. Confocal laser scanning microscopy using the fluorescent marker FM 4-64 confirmed the increasing number of vesicles induced by ALA in the pollen tubes. Ultrastructural analysis of pollen tubes treated by ALA with exogenous CaCl2 or/ and EGTA not only indicated that a Ca2+ signal plays a crucial role in ALA-mediated increase of vesicles, but also suggested that destruction of endoplasmic reticulum and Golgi bodies is one of the vesicle sources. Confocal microscopy of FM 4-64 further demonstrated that Ca2+ signal involves in ALA-mediated increase of vesicles also through promoting endocytosis and inhibiting exocytosis. In addition, treatment of taxol, a microtubule stabilizer, partially rescued the effects of ALA on pollen tube growth and FM 4-64 staining, indicating that microtubules are involved in ALA-regulated pollen tube growth. Taken together, we conclude that ALA-decreased Ca2+ disrupts endoplasmic reticulum and Golgi apparatus, enhances endocytosis, suppresses exocytosis, impairs vesicle transport, and finally leads to vesicle accumulation and abnormality of pollen tubes. Our findings provide new information on how ALA regulates pollen tube growth.

Transmission electron microscopic and immunohistochemical observations of resting follicles of feathers in chicken show massive cell degeneration

The molting cycle of feathers includes an anagen (growth) stage, a likely catagen stage where the feather follicles degenerate, and a resting stage where fully grown feathers remain in their follicles and are functional before molting. However, the cytological changes involved in the resting and molting stages are poorly known, so the results of an ultrastructural analysis of these processes in adult chick feathers are presented here. The study showed that the dermal papilla shrinks, and numerous cells present increased heterochromatin and free collagen fibrils in the extracellular matrix. Degeneration of the germinal epithelium of the follicle-the papillary collar-occurs with an initial substantial contraction of cells followed by an increase in heterochromatin, vesicle and lipid accumulation, and membrane and organelle degeneration. Desmosomes are still present between degenerating epithelial cells, but ribosomes and tonofilaments disappear. This suggests that cell necrosis initially proceeds as a major contraction resembling apoptosis-a process termed necroptosis, which was previously also shown to occur during the formation of barbs and barbules in mature down and pennaceous feathers. This study suggests that, aside from apoptosis, the collar epithelium degenerates due to external factors, in particular the retraction of blood vessels supplying the dermal papilla. In contrast, revascularization of the dermal papilla triggers a new phase of feather growth (anagen).

Accumulation of Dense Core Vesicles in Hippocampal Synapses Following Chronic Inactivity.

The morphology and function of neuronal synapses are regulated by neural activity, as manifested in activity-dependent synapse maturation and various forms of synaptic plasticity. Here we employed cryo-electron tomography (cryo-ET) to visualize synaptic ultrastructure in cultured hippocampal neurons and investigated changes in subcellular features in response to chronic inactivity, a paradigm often used for the induction of homeostatic synaptic plasticity. We observed a more than 2-fold increase in the mean number of dense core vesicles (DCVs) in the presynaptic compartment of excitatory synapses and an almost 20-fold increase in the number of DCVs in the presynaptic compartment of inhibitory synapses after 2 days treatment with the voltage-gated sodium channel blocker tetrodotoxin (TTX). Short-term treatment with TTX and the N-methyl-D-aspartate receptor (NMDAR) antagonist amino-5-phosphonovaleric acid (AP5) caused a 3-fold increase in the number of DCVs within 100 nm of the active zone area in excitatory synapses but had no significant effects on the overall number of DCVs. In contrast, there were very few DCVs in the postsynaptic compartments of both synapse types under all conditions. These results are consistent with a role for presynaptic DCVs in activity-dependent synapse maturation. We speculate that these accumulated DCVs can be released upon reactivation and may contribute to homeostatic metaplasticity.

A Novel 2-Methoxyestradiol Analogue Is Responsible for Vesicle Disruption and Lysosome Aggregation in Breast Cancer Cells

Background: 2-Methoxyestradiol (2ME2) is an endogenous metabolite of 17-β-estradiol with anti-proliferative and anti-angiogenic properties. Due to 2ME2’s rapid metabolism and low oral bioavailability in in vivo settings, 2ME2 analogues have been designed to alleviate these issues. One of these compounds is 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). A previous work alluded to the ability of ESE-16 to induce autophagic cell death. Therefore, we investigated the mode of action of ESE-16 by studying its effects on autophagy, vesicle formation, and lysosomal organisation. Summary: Vesicle formation and autophagy induction were analysed by transmission electron microscopy (TEM), monodansylcadaverine (MDC) staining and Lysotracker staining, while autophagosome turnover was analysed using microtubule-associated protein 1A/1B-light chain 3 (LC3 lipidation) analysis. MDC staining of acidic vesicles revealed an increase both in the number and size of vesicles after ESE-16 exposure. This was confirmed by TEM. Lysotracker staining indicated an increase in the size of lysosomes, as well as changes in their distribution within the cell. However, autophagy was not induced, since LC3 lipidation did not increase after exposure to ESE-16. Key ­Messages: This study showed that ESE-16 exposure leads to the aggregation of acidic vesicles, identified as lysosomes, not accompanied by an induction of autophagy. Therefore, ESE-16 disrupts normal endocytic vesicle maturation likely through the inhibition of the microtubule function.