Movement Of Vesicles Research Articles

Motility of Enzyme-Powered Vesicles.

Autonomous nanovehicles powered by energy derived from chemical catalysis have potential applications as active delivery agents. For in vivo applications, it is necessary that the engine and its fuel, as well as the chassis itself, be biocompatible. Enzyme molecules have been shown to display enhanced motility through substrate turnover and are attractive candidates as engines; phospholipid vesicles are biocompatible and can serve as cargo containers. Herein, we describe the autonomous movement of vesicles with membrane-bound enzymes in the presence of the substrate. We find that the motility of the vesicles increases with increasing enzymatic turnover rate. The enhanced diffusion of these enzyme-powered systems was further substantiated in real time by tracking the motion of the vesicles using optical microscopy. The membrane-bound protocells that move by transducing chemical energy into mechanical motion serve as models for motile living cells and are key to the elucidation of the fundamental mechanisms governing active membrane dynamics and cellular movement.

11. TRANSCRIPTOME ANALYSIS AND TIME-LAPSE OBSERVATION OF A CASE WITH REPEATED MULTIPLE PRONUCLEI AFTER ICSI

Introduction Pronucleus (PN) formation is a process by which the parental chromosome complements unite and engender the zygotic genome. During fertilization, the maternal PN is pulled toward the male counterpart, and the 2PN are juxtaposed and positioned in the central or paracentral region of the cytoplasm (Terada et al., 2010). More than 2PN can sometimes be observed, which is generally assumed to be due to either a polyspermic penetration or to the failure of a second polar body extrusion, especially in the case of 3PN (Rosenbusch et al., 2010). However, for oocytes with >3PN, the reason of their incidence remains unclear. The aim of the study was to identify the cause of repeated multipronuclei (MPN) formation in zygotes of a patient after intracytoplasmic sperm injection (ICSI). Material and Methods This is a case study of a patient with repeated MPN formation after five ICSI cycles (four in other clinics and one in Istanbul Memorial Hospital). Time-lapse monitoring of PN formation was carried out. Embryos developed from seven MPN zygotes of the sixth cycle were analyzed by FISH with the informed consent of the patient. Six MPN day-3 embryos and two surplus day-3 embryos for which the couples did not accept freezing were collected with informed consent from the patient and two couples, respectively. They were analyzed by whole genome expression analysis (Clariom™ D Assay, ThermoFisher Scientific). Results The time-lapse evaluation of the patient's 17 oocytes showed 9 MPN zygotes, one 3PN zygote, two unfertilized oocytes and three normally fertilized zygotes. The paternal pronuclei was of normal size and 7-9 micropronuclei appeared beneath the second polar body and migrated to the center of the cytoplasm. The ensuing cleavages were unequal and gave rise to a high rate of fragmentation. All MPN and the 3PN embryos arrested their development on day-3. Seven MPN embryos were analyzed by FISH and 1-20 micronuclei were observed in each blastomere. Six embryos were diagnosed as being chaotic aneuploid and one as being monosomy18 and nullisomy13. Transcriptomes of six MPN arrested day-3 embryos and two surplus 2PN good quality day-3 embryos were compared. Three pathways involving the AXIN1 gene were found to be significantly affected: Wnt signaling pathway and pluripotency, embryonic stem cell pluripotency pathways and the apoptosis-related network due to altered Notch3 in ovarian cancer. The chemokine signaling pathway (CCL19, FGR, PREX1 genes) known to be involved in the regulation of the actin cytoskeleton, which has a role in cell division, vesicle and organelle movement and interestingly documented to be disrupted in patients with premature ovarian failure was also found to be involved. Conclusions Several candidate coding genes involved in cell cycle, pluripotency of stem cells and actin cytoskeleton pathways have been identified as a new cause of MPN formation and infertility. However, transcriptional data from six MPN embryos can only provide clues for further investigation. Future studies, including whole exon sequencing of candidate genes of the patient, her family and other patients with MPN zygotes might help in identifying the exact mechanism of this phenomenon.

Plastic Deformation and Fragmentation of Strained Actin Filaments

The assembly of actin filaments and filament networks generate forces that drive cell and vesicle movement. These structures and the comprising actin filaments must be mechanically stable to sustain these forces and maintain their structural integrity. Filaments in these dynamic structures must also be disassembled to recycle and replenish the pool of actin monomers available for polymerization. Actin-severing proteins such as cofilin and contractile myosin motor proteins fragment these nominally stable structures. We developed a mesoscopic-length-scale actin filament model to investigate force-induced filament fragmentation. We show that fragmentation in our model occurs at curvatures similar to previous measurements of fragmentation within (cofil)actin and actin-cofilactin boundaries. Boundaries between bare and cofilin-decorated segments are brittle and fragment at small bending and twisting deformations. Extending filaments disperses strain uniformly over subunit interfaces, and filaments fragment with no detectable partial rupture or plastic deformation. In contrast, bending or twisting filaments imposes nonuniform interface strain and leads to partial interface rupture, accelerating filament fragmentation. As a result, the rupture force under compressive loads is an order of magnitude lower than under tensile loads. Partial interface rupture may be a primary mechanism of accelerating actin filament fragmentation by other actin-destabilizing proteins.

1773-P: Beta-Catenin Is a Key Player in Insulin-Stimulated Skeletal Muscle Glucose Transport

Recent work has identified β-catenin as a potential regulator of vesicle trafficking within various tissues including synapses, pancreatic β-cells, and adipocytes. The trafficking of GLUT4 vesicles to the cell membrane is essential for insulin-stimulated glucose uptake. Here we provide evidence for a role of β-catenin in regulating skeletal muscle glucose transport. In in vivo models, β-catenin is sensitive to insulin, which through PI3 kinase and Akt triggers phosphorylation of β-catenin at serine 552 (S552). Substituting S552 to alanine impairs glucose transport in L6-GLUT4-myc cells suggesting S552 is key in β-catenin-regulated glucose transport. Furthermore, inhibition of β-catenin in ex vivo models and cell culture impairs insulin-stimulated glucose transport. Using transcriptional inhibitors we demonstrate that β-catenin’s regulation of glucose transport is at least in part independent of its role in gene regulation. Finally, we investigated whether diet induced insulin-resistance alters insulin stimulated β-cateninS552 phosphorylation in vivo. Based on β-catenin’s structure and role in other cellular processes we believe that β-catenin regulates glucose transport via interactions with cytoskeletal proteins which ultimately regulate vesicle movement. Disclosure S.W.C. Masson: None. P.R. Shepherd: None. T.L. Merry: None. Funding Royal Society of New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery

Novel exc Genes Involved in Formation of the Tubular Excretory Canals of Caenorhabditis elegans.

Regulation of luminal diameter is critical to the function of small single-celled tubes, of which the seamless tubular excretory canals of Caenorhabditis elegans provide a tractable genetic model. Mutations in several sets of genes exhibit the Exc phenotype, in which canal luminal growth is visibly altered. Here, a focused reverse genomic screen of genes highly expressed in the canals found 18 genes that significantly affect luminal outgrowth or diameter. These genes encode novel proteins as well as highly conserved proteins involved in processes including gene expression, cytoskeletal regulation, and vesicular and transmembrane transport. In addition, two genes act as suppressors on a pathway of conserved genes whose products mediate vesicle movement from early to recycling endosomes. The results provide new tools for understanding the integration of cytoplasmic structure and physiology in forming and maintaining the narrow diameter of single-cell tubules.

Electrophoretic transport and dynamic deformation of bio-vesicles.

Study of the deformation dynamics of cells and other sub-micron vesicles, such as virus and neurotransmitter vesicles are necessary to understand their functional properties. This mechanical characterization can be done by submerging the vesicle in a fluid medium and deforming it with a controlled electric field, which is known as electrodeformation. Electrodeformation of biological and artificial lipid vesicles is directly influenced by the vesicle and surrounding media properties and geometric factors. The problem is compounded when the vesicle is naturally charged, which creates electrophoretic forcing on the vesicle membrane. We studied the electrodeformation and transport of charged vesicles immersed in a fluid media under the influence of a DC electric field. The electric field and fluid-solid interactions are modeled using a hybrid immersed interface-immersed boundary technique. Model results are verified with experimental observations for electric field driven translocation of a virus through a nanopore sensor. Our modeling results show interesting changes in deformation behavior with changing electrical properties of the vesicle and the surrounding media. Vesicle movement due to electrophoresis can also be characterized by the change in local conductivity, which can serve as a potential sensing mechanism for electrodeformation experiments in solid-state nanopore setups.

Turnip Mosaic Virus Components Are Released into the Extracellular Space by Vesicles in Infected Leaves.

Turnip mosaic virus (TuMV) reorganizes the endomembrane system of the infected cell to generate endoplasmic-reticulum-derived motile vesicles containing viral replication complexes. The membrane-associated viral protein 6K2 plays a key role in the formation of these vesicles. Using confocal microscopy, we observed that this viral protein, a marker for viral replication complexes, localized in the extracellular space of infected Nicotiana benthamiana leaves. Previously, we showed that viral RNA is associated with multivesicular bodies (MVBs). Here, using transmission electron microscopy, we observed the proliferation of MVBs during infection and their fusion with the plasma membrane that resulted in the release of their intraluminal vesicles in the extracellular space. Immunogold labeling with a monoclonal antibody that recognizes double-stranded RNA indicated that the released vesicles contained viral RNA. Focused ion beam-extreme high-resolution scanning electron microscopy was used to generate a three-dimensional image that showed extracellular vesicles in the cell wall. The presence of TuMV proteins in the extracellular space was confirmed by proteomic analysis of purified extracellular vesicles from N benthamiana and Arabidopsis (Arabidopsis thaliana). Host proteins involved in biotic defense and in interorganelle vesicular exchange were also detected. The association of extracellular vesicles with viral proteins and RNA emphasizes the implication of the plant extracellular space in viral infection.

MiRNA deregulation can contribute to breakdown of MHC I class antigen processing and presenting machinery in breast cancer cells

Background: To attack tumors, cytotoxic CD8+ T cells need to recognize tumor antigen peptides loaded onto major histocompatibility complex (MHC) class I molecules. Antigen processing and presentation is multistep process that includes proteasome-dependent and independent cleavage of cytosolic proteins, transport of derived peptides into the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAPs), peptide trimming by ER aminopeptidases (ERAPs), peptide loading onto MHC molecules with the help of calnexin, calreticulin, protein disulfide-isomerase A3 and tapasin, movement of vesicles with MHC-peptide complex via the Golgi apparatus to the cell surface. Being one of the main immune escape pathways, defects in this machinery are frequently observed in various tumors and correlate with tumor grade and aggressiveness. This research aims to identify how the tumor-related abnormalities in microRNA (miRNA) expression pattern can disrupt antigen processing and presentation in breast cancer cells.

The Potential of Combining Tubulin-Targeting Anticancer Therapeutics and Immune Therapy.

Cancer immune therapy has recently shown tremendous promise to combat many different cancers. The microtubule is a well-defined and very effective cancer therapeutic target. Interestingly, several lines of evidence now suggest that microtubules are intimately connected to the body’s immune responses. This raises the possibility that the combination of microtubule inhibitors and immune therapy can be a highly effective option for cancer treatments. However, our understanding on this potentially important aspect is still very limited, due in part to the multifaceted nature of microtubule functions. Microtubules are not only involved in maintaining cell morphology, but also a variety of cellular processes, including the movement of secretory vesicles and organelles, intracellular macromolecular assembly, signaling pathways, and cell division. Microtubule inhibitors may be subdivided into two classes: Anti-depolymerization agents such as the taxane family, and anti-polymerization agents such as colchicine and vinka alkaloids. These two different classes may have different effects on immune cell subtypes. Anti-depolymerization agents can not only induce NK cells, but also appear to inhibit T regulatory (Treg) cells. However, different inhibitors may have different functions even among the same class. For example, the doxetaxel anti-depolymerization agent up-regulates cytotoxic T cells, while paclitaxel down-regulates them. Certain anti-polymerization agents such as colchicine appear to down-regulate most immune cell types, while inducing dendritic cell maturation and increasing M1 macrophage population. In contrast, the vinblastine anti-polymerization agent activates many of these cell types, albeit down-regulating Treg cells. In this review, we focus on the various effects of tubulin inhibitors on the activities of the body’s immune system, in the hope of paving the way to develop an effective cancer therapy by combining tubulin-targeting anticancer agents and immune therapy.

CLIP-170 spatially modulates receptor tyrosine kinase recycling to coordinate cell migration.

Endocytic sorting of activated receptor tyrosine kinases (RTKs), alternating between recycling and degradative processes, controls signal duration, location and surface complement of RTKs. The microtubule (MT) plus‐end tracking proteins (+TIPs) play essential roles in various cellular activities including translocation of intracellular cargo. However, mechanisms through which RTKs recycle back to the plasma membrane following internalization in response to ligand remain poorly understood. We report that net outward‐directed movement of endocytic vesicles containing the hepatocyte growth factor (HGF) Met RTK, requires recruitment of the +TIP, CLIP‐170, as well as the association of CLIP‐170 to MT plus‐ends. In response to HGF, entry of Met into Rab4‐positive endosomes results in Golgi‐localized γ‐ear‐containing Arf‐binding protein 3 (GGA3) and CLIP‐170 recruitment to an activated Met RTK complex. We conclude that CLIP‐170 co‐ordinates the recycling and the transport of Met‐positive endocytic vesicles to plus‐ends of MTs towards the cell cortex, including the plasma membrane and the lamellipodia, thereby promoting cell migration.

Dynamic single-vesicle tracking of cell-bound membrane vesicles on resting, activated, and cytoskeleton-disrupted cells

The composition, structure, production, motion, fate, and functions of cell-bound membrane vesicles pre-existing in the plasma membrane of cells are poorly understood. Here, single-vesicle tracking of individual cell-bound membrane vesicles in the plasma membrane of endothelial cells treated with or without various reagents was performed to investigate the motion of cell-bound membrane vesicles. The efficacy of each of these reagents was confirmed prior to single-vesicle tracking. Via single-vesicle tracking, we found that oxLDL, TNF-α, and VEGFα significantly increased the average number of cell-bound membrane vesicles per cell, implying that cell activation by oxLDL, TNF-α, and VEGFα could trigger the production of cell-bound membrane vesicles. It was also found that oxLDL, TNF-α, VEGFα, LPS, and MβCD but not LDL could significantly affect the motion speed of cell-bound membrane whereas none of them could significantly influence the displacement (moving range) of cell-bound membrane vesicles. The single-vesicle tracking further revealed that the average number of cell-bound membrane vesicles per cell and the mean speed/displacement of individual cell-bound membrane vesicles could be dramatically altered by the cytoskeleton-disrupting reagents (cytochalasin D and nocodazole). The data imply that the production and movement of cell-bound membrane vesicles are probably controlled by intracellular cytoskeletons and capable of being affected by multiple conditions e.g. cell activation, membrane fluidity alteration, and others.

Movement of giant lipid vesicles induced by millimeter wave radiation change when they contain magnetic nanoparticles.

Superparamagnetic iron oxide nanoparticles are used in a rapidly expanding number of research and practical applications in biotechnology and biomedicine. Recent developments in iron oxide nanoparticle design and understanding of nanoparticle membrane interactions have led to applications in magnetically triggered, liposome delivery vehicles with controlled structure. Here we study the effect of external physical stimuli-such as millimeter wave radiation-on the induced movement of giant lipid vesicles in suspension containing or not containing iron oxide maghemite (γ-Fe2O3) nanoparticles (MNPs). To increase our understanding of this phenomenon, we used a new microscope image-based analysis to reveal millimeter wave (MMW)-induced effects on the movement of the vesicles. We found that in the lipid vesicles not containing MNPs, an exposure to MMW induced collective reorientation of vesicle motion occurring at the onset of MMW switch "on." Instead, no marked changes in the movements of lipid vesicles containing MNPs were observed at the onset of first MMW switch on, but, importantly, by examining the course followed; once the vesicles are already irradiated, a directional motion of vesicles was induced. The latter vesicles were characterized by a planar motion, absence of gravitational effects, and having trajectories spanning a range of deflection angles narrower than vesicles not containing MNPs. An explanation for this observed delayed response could be attributed to the possible interaction of MNPs with components of lipid membrane that, influencing, e.g., phospholipids density and membrane stiffening, ultimately leads to change vesicle movement.

Turnip Mosaic Virus Uses the SNARE Protein VTI11 in an Unconventional Route for Replication Vesicle Trafficking.

Infection of plant cells by RNA viruses leads to the generation of organelle-like subcellular structures that contain the viral replication complex. During Turnip mosaic virus (TuMV) infection of Nicotiana benthamiana, the viral membrane protein 6K2 plays a key role in the release of motile replication vesicles from the host endoplasmic reticulum (ER). Here, we demonstrate that 6K2 contains a GxxxG motif within its predicted transmembrane domain that is vital for TuMV infection. Replacement of the Gly with Val within this motif inhibited virus production, and this was due to a relocation of the viral protein to the Golgi apparatus and the plasma membrane. This indicated that passage of 6K2 through the Golgi apparatus is a dead-end avenue for virus infection. Impairing the fusion of transport vesicles between the ER and the Golgi apparatus by overexpression of the SNARE Sec22 protein resulted in enhanced intercellular virus movement. Likewise, expression of nonfunctional, Golgi-located synaptotagmin during infection enhanced TuMV intercellular movement. 6K2 copurified with VTI11, a prevacuolar compartment SNARE protein. An Arabidopsis thaliana vti11 mutant was completely resistant to TuMV infection. We conclude that TuMV replication vesicles bypass the Golgi apparatus and take an unconventional pathway that may involve prevacuolar compartments/multivesicular bodies for virus infection.

Abstract B57: Microtubule dynamics regulates mammary gland morphogenesis and tumorigenesis via stathmin

Abstract Microtubule (MT) dynamics is essential to provide a correct apico-basal polarization of epithelial cells, by regulating the movement of vesicles and proteins within the cell and by ensuring a correct orientation of the mitotic spindle during cell division. The dynamics of MT is governed by the activity of MT-stabilizing and destabilizing proteins, generally known as MT-acting proteins. Little is known about the role of the MT-dynamics and of MT-acting proteins during development of the mammary gland, a dynamic organ that displays a complex and finely organized apico-basal architecture. Stathmin, a MT-destabilizing protein, is often overexpressed in breast cancer (BC). By studying knockout (STM KO) mice we observed that STM KO females were unable to nurse their offspring. This phenotype was accompanied by defective development of the mammary gland, particularly evident during pregnancy and postpartum phases. Our results showed that stathmin absence mainly impinged on two key features of the mammary epithelial organization, polarity and proliferation. In vitro, normal mammary epithelial cells silenced for stathmin were not able to form organized acini in 3D-matrices. In vivo, when stathmin was KO, many molecules involved in mammary gland functions were not properly localized, indicating a disruption of the apico-basal polarity. Furthermore, loss of stathmin strongly decreased proliferation and induced disoriented positioning of the mitotic spindle in dividing epithelial cells of the developing alveolus, preventing the correct orientation of the daughter cells. Loss of polarity and deregulated proliferation are tightly linked to tumorigenesis. We therefore decided to examine the role of stathmin also in the context of HER2-driven tumorigenesis. To this end, we used a transgenic mouse model expressing a constitutively active form of HER2 (Δ16HER2), which is an alternatively spliced, very aggressive form of HER2, and intercrossed them with STM WT and KO mice. We analyzed the role of stathmin in both early and late stages of tumorigenesis. STM KO mice displayed a significantly reduced number of preneoplastic foci and this phenotype was maintained also in late stages of tumorigenesis. In WT mice, preneoplastic lesions expressed high level of stathmin compared with normal tissue, supporting the idea that stathmin played an active role in tumor initiation. Analysis of proliferation in tumors revealed that loss of stathmin strongly decreased the mitotic rate of Δ16HER2-transformed cells. Furthermore, by syngeneic injection of tumor cells in recipient mice of the two different genotypes, we could assess that stathmin plays a cell-autonomous function in tumor initiation. Our study has shed new light on the field of mammary gland development and of breast cancer biology. Collectively, our data indicate that loss of stathmin by altering the dynamics of microtubules causes a profound disorganization of the normal mammary gland architecture and a delay in initiation of the tumorigenic process. We have uncovered new functions of stathmin that could have important implications in BC, also considering that several microtubule-acting drugs are currently employed in the therapy of BC patients. Citation Format: Ilenia Segatto, Mara De Marco Zompit, Gian Luca Rampioni Vinciguerra, Giorgia Mungo, Sara D’Andrea, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. Microtubule dynamics regulates mammary gland morphogenesis and tumorigenesis via stathmin [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B57.

Moving Droplets in 3D Using Light.

The emulation of the complex cellular and bacterial vesicles used to transport materials through fluids has the potential to add revolutionary capabilities to fluidic platforms. Although a number of artificial motile vesicles or microdroplets have been demonstrated previously, control over their movement in liquid in 3D has not been achieved. Here it is shown that by adding a chemical "fuel," a photoactive material, to the droplet, it can be moved in any direction (3D) in water using simple light sources without the need for additives in the water. The droplets can be made up of a range of solvents and move with speeds as high as 10.4 mm s-1 toward or away from the irradiation source as a result of a light-induced isothermal change in interfacial tension (Marangoni flow). It is further demonstrated that more complex functions can be accomplished by merging a photoactive droplet with a droplet carrying a "cargo" and moving the new larger droplet to a "reactor" droplet where the cargo undergoes a chemical reaction. The control and versatility of this light-activated, motile droplet system will open up new possibilities for fluidic chemical transport and applications.

The Escherichia coli Type III Secretion System 2 Has a Global Effect on Cell Surface.

ABSTRACTMany strains of Escherichia coli carry a 29,250-bp ETT2 pathogenicity island (PAI), which includes genes predicted to encode type III secretion system (T3SS) components. Because it is similar to the Salmonella pathogenicity island 1 (SPI-1) system, encoding a T3SS in Salmonella enterica, it was assumed that ETT2 also encodes a secretion system injecting effectors into host cells. This assumption was checked in E. coli serotype O2—associated with urinary tract infections and septicemia—which has an intact ETT2 gene cluster, in contrast to most strains in which this cluster carries deletions and mutations. A proteomic search did not reveal any putative secreted effector. Instead, the majority of the secreted proteins were identified as flagellar proteins. A deletion of the ETT2 gene cluster significantly reduced the secretion of flagellar proteins, resulting in reduced motility. There was also a significant reduction in the transcriptional level of flagellar genes, indicating that ETT2 affects the synthesis, rather than secretion, of flagellar proteins. The ETT2 deletion also resulted in additional major changes in secretion of fimbrial proteins and cell surface proteins, resulting in relative resistance to detergents and hydrophobic antibiotics (novobiocin), secretion of large amounts of outer membrane vesicles (OMVs), and altered multicellular behavior. Most important, the ETT2 deletion mutants were sensitive to serum. These major changes indicate that the ETT2 gene cluster has a global effect on cell surface and physiology, which is especially important for pathogenicity, as it contributes to the ability of the bacteria to survive serum and cause sepsis.

Abstract 1460: Stathmin regulates mammary gland morphogenesis and tumorigenesis

Abstract Microtubule (MT) dynamics is essential to provide a correct apico-basal polarization of epithelial cells, by regulating the movement of vesicles and proteins within the cell and by ensuring a correct orientation of the mitotic spindle during cell division. Little is known about the role of the MT-dynamics and of MT-regulating proteins during development of the mammary gland, a dynamic organ that displays a complex and finely organized apico-basal architecture. Stathmin, a MT-destabilizing protein, is often overexpressed in breast cancer (BC). By studying knockout (STM KO) mice we observed that STM KO females were unable to nurse their offspring. This phenotype was accompanied by defective development of the mammary gland, particularly evident during pregnancy and post-partum phases. Our results showed that stathmin absence mainly impinged on two key features of the mammary epithelial organization, polarity and proliferation. In vitro, normal mammary epithelial cells silenced for stathmin were not able to form organized acini in 3D-matrices. In vivo, when stathmin was KO, many molecules involved in mammary gland functions were not properly localized, indicating a disruption of the apico-basal polarity. Furthermore, loss of stathmin strongly decreased proliferation and induced disoriented positioning of the mitotic spindle in dividing epithelial cells, preventing the correct orientation of the daughter cells. Loss of polarity and deregulated proliferation are tightly linked to tumorigenesis. We therefore examined the role of stathmin also in the context of HER2-driven tumorigenesis. To this end, we used a transgenic mouse model expressing a constitutively active form of HER2 (Δ16HER2), which is an alternatively spliced, very aggressive form of HER2, and intercrossed them with STM WT and KO mice. We analyzed the role of stathmin in both early and late stages of tumorigenesis. STM KO mice displayed a significantly reduced number of pre-neoplastic foci and this phenotype was maintained also in late stages of tumorigenesis. In WT mice, pre-neoplastic lesions expressed high level of stathmin compared with normal tissue supporting the idea that stathmin played an active role in tumor initiation. Analysis of proliferation in tumors revealed that loss of stathmin strongly decreased the mitotic rate of Δ16HER2-transformed cells. Furthermore, we assessed that stathmin plays a cell autonomous function in tumor initiation. Our study has shed new light in the field of mammary gland development and of breast cancer biology. Collectively, our data indicate that loss of stathmin by altering the dynamics of microtubules, causes a profound disorganization of the normal mammary gland architecture and a delay in initiation of the tumorigenic process. We have uncovered new functions of stathmin that could have important implications in BC, also in consideration that several microtubules acting drugs are currently employed in the therapy of BC patients Citation Format: Ilenia Segatto, Mara De Marco Zompit, Gian Luca Rampioni Vinciguerra, Francesca Citron, Sara D'Andrea, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. Stathmin regulates mammary gland morphogenesis and tumorigenesis [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 1460.

Regulation of KIF1A-Driven Dense Core Vesicle Transport: Ca2+/CaM Controls DCV Binding and Liprin-α/TANC2 Recruits DCVs to Postsynaptic Sites

SummaryTight regulation of neuronal transport allows for cargo binding and release at specific cellular locations. The mechanisms by which motor proteins are loaded on vesicles and how cargoes are captured at appropriate sites remain unclear. To better understand how KIF1A-driven dense core vesicle (DCV) transport is regulated, we identified the KIF1A interactome and focused on three binding partners, the calcium binding protein calmodulin (CaM) and two synaptic scaffolding proteins: liprin-α and TANC2. We showed that calcium, acting via CaM, enhances KIF1A binding to DCVs and increases vesicle motility. In contrast, liprin-α and TANC2 are not part of the KIF1A-cargo complex but capture DCVs at dendritic spines. Furthermore, we found that specific TANC2 mutations—reported in patients with different neuropsychiatric disorders—abolish the interaction with KIF1A. We propose a model in which Ca2+/CaM regulates cargo binding and liprin-α and TANC2 recruit KIF1A-transported vesicles.

Saturated Fatty Acids Impair Organellar Trafficking in Dorsal Root Ganglion (DRG) Sensory Neurons

Diabetic neuropathy (DN) is a highly prevalent complication of type 2 diabetes that is associated with dyslipidemia and causes length-dependent sensory nerve damage in the lower extremities. The primary sensory neurons, dorsal root ganglion (DRG) neurons, extend bundles of axons into the peripheral nerves and transmit sensory information from the limbs. These neurons require a constant supply of mitochondrial derived energy maintained by trafficking of the mitochondria throughout the length of the DRG axon. We have shown that dyslipidemia-associated saturated fatty acid (FA) palmitate impairs mitochondrial trafficking whereas unsaturated FA oleate prevents this impairment. In the current study, we assessed whether FAs alter trafficking of synaptic vesicles in DRG axons to determine whether palmitate impairs mitochondrial trafficking specifically or global organellar transport. Primary DRG neurons were treated with physiological concentrations of FAs ranging from 31.25-250 µM saturated FA palmitate, unsaturated FA oleate, and oleate/palmitate mixtures. Mitochondria and synaptic vesicles showed a significant dose-dependent reduction in motility with increasing concentrations of saturated FA palmitate. However, mitochondria and synaptic vesicles retained motility with treatments of unsaturated FA oleate compared to the controls. Similarly, palmitate induced a trending decrease in velocity of bi-directional motile mitochondria and synaptic vesicles, whereas oleate treatments did not alter mitochondrial and synaptic vesicle velocity. Finally, co-incubation of oleate prevents palmitate-induced inhibition of mitochondrial and synaptic vesicle trafficking. These results suggest that high concentrations of saturated FAs lead to the dysfunction of global organellar transport throughout the DRG axon associated with the development and progression of DN. Disclosure G. LoGrasso: None. A. Rumora: None. J.A. Haidar: None. S.I. Lentz: None. E.L. Feldman: None.

Numerical method for vesicle movement analysis in a complex cytoskeleton network

The detection of the precise movement of a vesicle during transport in a live cell provides key information for the intracellular delivery process. Here we report a novel numerical method for analyzing three-dimensional vesicle movement. Since the vesicle moves along a linear cytoskeleton during the active transport, our method first detects the orientation and position of the cytoskeleton as a linear section based on angle correlation and linear regression, after noise reduction. Then, the precise vesicle movement is calculated using vector analysis, in terms of rotation angle and translational displacement. Using this method, various vesicle trajectories obtained via high spatiotemporal resolution microscopy can be understood..