Cellular Mechanisms Underlying the Evasion of Detachment-Induced Cell Death in Inflammatory Breast Cancer Cells

Brittany Angarola Advisor: Zachary Schafer, Dept. of Biological Sciences

Inflammatory Breast Cancer (IBC) is a very rare and aggressive form of breast cancer that results from cancerous epithelial cells that invade and block the lymphatic vessels. Because the cancerous cells have already migrated from the epithelium into the lymphatic system, these cells are inherently metastatic and have the ability to survive in the absence of attachment to the extracellular matrix (ECM). Non-cancerous epithelial cells require ECM attachment for survival, however IBC cells are able to overcome both detachment-induced cell death and metabolic stress in order to survive in the lymphatic vessels. Here we demonstrate that both the SUM149 and the ErbB2-overexpressing SUM190 cell lines derived from primary IBC tumors have acquired mechanisms to aid in their survival in detached environments. This is evidenced by lower levels of caspase activation, which allow both cell lines to avoid anoikis (detachment- induced apoptosis). While the SUM149 cells appear to maintain survival signaling via the MAPK pathway to avoid cell death, the SUM190 cells more effectively evade cell death by blocking detachment-induced induction of caspase-3. Additionally, both the SUM149 cells and the SUM190 cells are able to overcome metabolic stress and rescue ATP levels in the detached state through phosphatidylinositol-3-OH kinase (PI3K) activation. This enhanced survival signaling upon detachment in IBC cells suggests there is a stabilization of receptor proteins, possibly implicating EGFR in SUM149 cells and overexpressed ErbB2 in SUM190 cells. These results show that IBC cells have capacity to alter both apoptotic and metabolic pathways to survive in detached environments. Further elucidation of these mechanisms may reveal new chemotherapeutic targets that permit the specific elimination of detached IBC cells within the lymphatic system.

Identifying the mechanism of exosome release; knockdown of Rab27a using siRNA in macrophage Raw Cells

Elaina Breznau Coauthor: Chris LeMaire Advisor: Jeffery Schorey Dept. Biological Sciences

Exosomes have recently been shown to function in intercellular signal transmission leading to tumor suppression and proinflammatroy response in different cell types (Raposo et al., 1996; Andre et al., 2004; Schorey and Bhatnagar 2008). The potential for using exosomes to direct the immune system or as a vaccine delivery method against infectious diseases is intriguing; however the precise mechanisms of exosome biogenesis remain elusive. In this study we aim to establish macrophage specific siRNA knockdowns of Rab27a, a protein implicated in exosome biogenesis, in order to characterize its role in the release of exosomes. Specifically, we hypothesize that decreasing Rab27a protein expression in 264.7 Raw cells will result in a significant decrease in the number of exosomes released from macrophages when compared to controls. Total RNA was purified from knockdown cells harvested 16, 24, and 48 hours post transfection and cell culture supernatant was collected for exosome quantification. Rab27a cDNA was quantified using qRT-PCR to determine the level of knockdown obtained for various treatment conditions and cell harvest times. Our results show that the best knockdown (53% and 64%) was obtained 24 hours post-transfection when using 7 μl and 3.5 μl siRNA respectively. Exosomes were purified from the culture supernatant of cells transfected with the siRNA and the amount of LAMP1 molecules present was determined by Western blot. LAMP1 is a protein found in high abundance on exosomes released from 264.7 Raw cells and differences in its concentration are predicted to correlate with the amount of exosomes. Our results indicate the amount of LAMP1 was not different between the samples suggesting that the knockdown of Rab27a did not modulate exosome release under the conditions tested. However, due to the low concentration of exosomes in the culture media, exact protein concentration of the different preparations could not be determined. Therefore a scaled up experiment is needed to obtain a sufficient amount of exosomes for accurate quantification.

Genetic relatedness and parasite burden in non-human primates in Bali, Indonesia

Adeline Dozois Coauthors: Kelly Lane Advisor: Hope Hollocher, Dept. of Biological Sciences

The importance of genetic relatedness in cross-species transmission of infectious disease is well- known, with closely related species being more likely to share similar pathogens. The link between phylogentics and disease transmission has been studied especially well in non-human primates, because their high genetic similarity to humans makes them a significant reservoir for diseases that affect human populations. However, it is unclear whether genetic relatedness facilitates the transmission of disease between primates at the intra-specific or intra-population level, in part because kinship relationships are largely unknown for wild primate populations. Using genetic markers, we examined relatedness in two large populations of long-tailed macaques (Macaca fascicularis) on the island of Bali, Indonesia. We then determined the fine- scale genetic structure within one of these populations by assigning individual macaques to discrete kin clusters. We used the program Structure, which identifies clusters of related individuals based on correlated allele frequencies. After identifying the most probable number of kin groups, we then compared clusters generated across multiple runs. Initial tests showed that individuals clustered into two discrete groups representing each of the two populations, consistent with previous studies demonstrating a large degree of genetic differentiation between the sites. In order to determine genetic differences at the pedigree level, we analyzed samples within each population separately. We then compared parasite species diversity and infection intensity in macaques and identified several parasite species which were associated with certain kin groups. These results suggest a potential genotypic basis for pathogen host-specificity within populations.

Role of a specific kinase in the regulation of clock resetting in the mammalian circadian clock

Kevin Flanagan Coauthor: Stuart N. Peirson, Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford Advisor: Giles Duffield, Dept. of Biological Sciences

Numerous physiological and behavioral processes are regulated by rhythmic gene and protein expression driven by cell-autonomous circadian clocks, regulated by the master clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The clock is reset by factors such as light through the transient induction or suppression of period 1 (per1) and period 2 (per2) transcripts. Higher per1 and per2 expression is believed to correspond with larger shifts in the circadian clock. One putative mechanism of per1 and per2 regulation is via a specific kinase, referred to as kinase X (KinX). KinX phosphorylation of transducer of regulated CREB activity (TORC) negatively regulates pCREB-specific gene transcription. As such, we hypothesized that KinX limits the light-induction of per1 and per2, thereby limiting the magnitude of subsequent circadian shifts. To test this hypothesis we used an in vitro model system of immortalized mouse embryonic fibroblasts (MEFs), which exhibit a circadian clock mechanism that can be reset by serum stimulation. Using qRT-PCR, we revealed that sik1 is induced by such a serum treatment, with expression peaking at 120 minutes, consistent with the kinx induction response in the SCN to light. Inhibition of KinX using a glycogen synthase kinase/cyclin-dependent kinase inhibitor, referred to as Inhibitor Y (InY), produced both an increase in the level and the duration of serum- induced per1 and per2 expression. Using MEFs derived from PER2::LUCIFERASE mice, we exposed cells to two serum treatments separated by 10 hours, the first to synchronize the cells, and the second to quantify a phase shift of the clock. The second serum treatment resulted in a 5.70 +/- 0.264 hour delay of the PER2:LUC rhythm. In InY treated cells, the phase shift increases to 9.00 +/- 0.41 hours. These data suggest that KinX limits the inducibility of per1 and per2 expression, thereby limiting the magnitude of phase shifts of the circadian clock. Future experiments will explore the contribution to KinX to light-resetting of the mouse SCN by treating light stimulated SCN ex vivo with InY, and subsequently monitoring the rhythm of per1:luciferase in cultured SCN.

Genetic and Environmental Influences on the “Third Generation” of the Karner Blue Butterfly

Arilene Gaxiola Advisor: Jessica Hellmann, Dept. of Biological Sciences

The Karner blue butterfly (Lycaeides melissa samuelis) is a federally listed endangered species that resides in seven of the 12 states in which it was historically found in the Great Lakes and Northeast region of the US. Despite habitat management, dramatic decreases in population have been observed in the last decades. This present study sought to determine whether Karner blue population declines may be attributed to climate change and the developmental life stage that is most vulnerable. The first generation begins as larvae in April and progresses through the butterfly life cycle, giving rise to the second generation adults in July. The eggs that are laid by these second generation females diapause overwinter. However, at least two captive rearing programs have observed hatching in some second generation eggs, though there is no documentation of this “third generation” hatching in the wild. In this study, we document the frequency of “third generation” hatching of eggs laid by wild-caught and captive reared females. We speculate that both genetic and environmental factors may be preventing the eggs from diapausing. We observed that hatching is female-specific, that is, greater than 95% of all the eggs laid by certain females hatched, which suggests that some genetic component is involved in early hatching. However, environmental cues, like daylight period, degree-days, and host plant quality and phenology can interact with these genotypes to trigger a break in diapause. Since both wild- caught and captive reared females were exposed to young, robust lupine plants and we observed differential hatching rates, host plant quality does not appear to be the major cause for third generation hatching. From studies examining diapause in other insects, degree days, which could be a consequence of predicted climate change, may be the most significant driving factor in triggering hatching in Karners. This third generation phenomenon may be significantly contributing to population declines resulting from what would normally be considered “overwintering mortality.” Host plant and nectar flowers required for survival may not be enough for these third generation butterflies, thus reducing the number of eggs that would overwinter to become the following year’s first generation.

The Effects of the Aggregation of Anchorage-Independent MCF-10A-ErbB2 cells on the PI3K and MAPK Pathways

Kelsea Hosoda Coauthor: Brittany Angarola Advisor: Zachary Schafer, Dept. of Biological Sciences

Metastasis is the process where cells separate from a primary tumor and form a secondary tumor and this mechanism is the leading cause of death in cancer patients. When normal epithelial cells detach from the extracellular matrix, deficient metabolism will result, followed by apoptosis or programmed cell death. This is not the case for cancer cells, which bypass apoptosis and propagate in the detached state. Cells that are forced to grow in a detached environment form aggregates. We have observed that MCF-10A cells (a normal mammary epithelial cell line) expressing ErbB2 form larger aggregates when grown in a detached state than wild-type MCF- 10A cells. Our previous studies have demonstrated that the overexpression of ErbB2 rescues the ATP deficient detached MCF-10A cells and increases cell viability by rectifying the metabolic defects and preventing apoptosis. In this study we have discovered that cell aggregation aids in the rescue of ATP and the prevention of apoptosis. When the PI3K and MAPK pathways were inhibited, there was less cell aggregation, ATP levels decreased and the induction of apoptosis increased. This was supported by the downregulation of the MAPK pathway, as seen by the increase in the amount of BIM and the decrease of phospho-ERK. The results from these experiments suggest potential pathways that my aid in cell aggregation and metastasis.

Leishmania major’s Effects on PU.1 and ETS-2 Involved in IL-12 Transcription

Adelaide Kamagaju Coauthors: Michelle Favila, Nicholas Geraci, Giordana Morales Advisor: Mary Ann McDowell, Dept. of Biological Sciences

Leishmania is a unicellular parasite transmitted by the bite of a phelbotomine sand fly. There are many species of Leishmania that can cause varying degrees of diseases in humans. Lesions form on spots where sand flies bite and these lesions go away overtime. Sometimes parasites flow in blood where they deposit and divide in the liver causing hepatomegaly. Previous research has suggested that Leishmania survives in the host by inhibiting interleukin-12 (IL-12), a cytokine necessary for an immune response to intracellular pathogens. There are several signal transduction pathways that occur in the cytoplasm leading to activation at the promoter sequence and subsequent transcription of IL-12. The signal transduction facilitates translocation of transcription factors from the cytoplasm to the nucleus where they can bind an ETS promoter sequence within the IL-12 promoter sequence. Transcription factors PU.1 and ETS-2 are inhibited during Leishmania infection causing IL-12 production to decrease and the effectiveness of fighting the infection to decrease as well. Nuclear and cytoplasmic extracts were used in Western blot analysis to determine whether PU.1 and ETS-2 were found in infected and uninfected samples. RAW 264.7 cells were infected with Leishmania major and stimulated with LPS, IFN-γ, or a combination of both LPS and IFN-γ, to activate different signaling pathways. ETS-2 and PU.1 were present in cytoplasmic and nuclear uninfected samples but not in infected samples. Our findings support our hypothesis that PU.1 and ETS-2 are inhibited during Leishmania infection. Our data suggests that Leishmania parasites may modulate signaling transduction pathways within host cells by regulating the translocation of PU.1 and ETS-2.

Does the transcription factor of CITED2 translocate into the nucleus during Leishmania infection?

Giordana Morales Advisor: Mary Ann McDowell, Dept. of Biological Sciences

Leishmaniasis is a vector-borne infectious disease transmitted by the bite of infected sandflies. The disease affects nearly 300 million people each year and is endemic to parts of the Middle East, Africa, and Central and South America. Leishmania spp. cause a spectrum of disease ranging from cutaneous (Leismania major) to visceral (L. donovani) leishmaniasis. At the cellular level, Leishmania major (L. major) primarily infect macrophages and evade intracellular destruction by regulating the host immune response. A typical immune response to infection involves an Interleukin-12 (IL-12) driven production of toxic nitric oxide. To persist within the cell, Leishmania spp inhibit IL-12 by inhibiting the ETS promoter within the IL-12 promoter region. Several transcription factors including ETS.2, PU.1, and CITED2, are known to regulate the ETS promoter. Using Western Blot analysis, we investigated whether L. major blocks transcription factor translocation of ETS.2, PU.1, and CITED2 into the nucleus to determine the cellular events that lead to inhibition of IL-12 following infection in macrophages.

Effect of light and dark adaptation on Op1 rhodopsin movement of Aedes aegypti

Angélica Pérez-Eguía, Coauthors: Michelle Whaley and Alexander Kiselev Advisor: Joseph O’Tousa, Dept. of Biological Sciences

Dengue and yellow fever are diseases transmitted by the Aedes aegypti mosquito. Rhodopsins, key mediators of process of visual perception, will broaden our knowledge on mosquito behavior such as biting, feeding, and mating. Our laboratory previously determined that AaOp1 is the major rhodopsin present in mosquitoes and that it is expressed in all the R1-R6 photoreceptor cells of the retina. My study investigates the movement of AaOp1 under light and dark conditions. Mosquitoes were treated to different hourly cycles of alternating dark and light conditions. The localization of Aaop1 was studied in whole mounts where the retinas were stained with an antibody to the C-terminus of Aaop1. Results indicate that AaOp1 is generally localized to the fused rhabdom in dark conditions and found in cytoplasmic vesicles and multivesicular bodies (MVBs) in the light. However, this pattern is not always consistent because we sometimes find cytoplasmic localization of AaOp1 in dark samples. A protein blot comparing A. aegypti heads at several of the five cycle light-dark treatment revealed that the amount of the Aaop1 protein is not dramatically different at each time point. When probed with an anti-ubiquitin antibody, however, a higher concentration of a 60kD complex known to be the opsin-ubiquitin complex was found in the light samples in which AaOp1 is present in the cytoplasm. This data suggest that there is a mechanism that alters the movement of Aaop1 and the sensitivity of the photoreceptor to different light conditions. This results show that even though A. aegypti mosquito is classified as diurnal which respect to their host seeking activity, their AaOp1photoreceptors are highly sensitized for performance in dim light conditions.

Generation of Drosophila Mutants to Study Light-driven Phosphorylation and Ubiquitination of Rh1 Rhodopsin

Ciara Reyes Advisor: Joseph O'Tousa, Dept. of Biological Sciences

Rhodopsins are light sensitive proteins that have a key role in visual transduction and photoreceptor maintenance. Several proteins are reported to regulate the function of rhodopsins including arrestins, rhodopsin kinase, rhodopsin phosphatase (RDG) and ubiquitin ligase. This regulation occurs through the posttranslational modification of rhodopsin such as phosphorylation and ubiquitination, and the particular modified rhodopsin states(s) may control the photoreceptor cell viability. For many G protein-coupled receptors it was shown that the phosphorylation initiates the ubiquitination process. It was the aim of this project to investigate the putative site of Rh1 rhodopsin phosphorylation and ubiquitination to better understand the role of this posttranslational modification in the regulation of photoreceptor function and turnover. To accomplish this goal, Drosophila mutants (S371E and K372R) were generated where putative site of phosphorylation (S371) and ubiquitination (K372) were altered to E and R residues, correspondingly. Using genetic crosses, K372R and S371E constructs were placed in an rdgC mutant background and subjected to DPP (deep dark pseudopupil) analysis to confirm the presence of rdgC by following the progression of light-driven retinal degeneration in living fly eyes. In addition all generated mutants were analyzed by Western blot analysis using anti- rhodopsin and anti-RDGC antibodies. The levels of mutated rhodopsins in all generated dark- reared flies were shown to be similar to the wild type animals. This establishes a good foundation to further investigate the light-driven characteristics of these mutants.

Investigation of mitotic phospho-dynein localization requirements and protein interactions

Matthew Sanchez Advisor: Kevin Vaughan, Dept. of Biological Sciences

Errors in mitotic division are major drivers in the formation of tumor cells. The minus end directed motor protein dynein is critical for proper progression through mitosis and differential phosphorylation along the dynein intermediate chain is responsible for determining dynein localization during mitosis. We have found that phosphorylation at a specific site on the dynein IC (Y130) causes dynein to localize directly to mitotic spindle poles, even in absence of proper microtubule formation. The spindle pole is composed of many proteins that must interact for proper spindle formation and identification of the kinase responsible for phosphorylation and recruitment of Y130 to spindle poles could aid in determining the role of Y130 dynein at spindle poles. Additionally, identification of Y130 binding partners could give needed insight into the interactions between specific spindle pole components. Using published proteomics information and available scientific literature, we compiled a list of candidate kinases most likely to cause Y130 localization. Small molecule inhibitors were then used to disrupt specific kinase activity in both NRK2 and HeLa cell lines. At this point, five candidate kinases have been tested using this method and ruled out due to the fact that inhibition of kinase activity did not cause Y130 delocalization. Immunoprecipitation of Y130 and subsequent silver staining resulted in multiple protein bands corresponding to potential binding partners. We are currently in the process of identifying these potential binding partner proteins. Further testing of kinases is also currently in progress, along with research into more candidates using new proteomics and mass spectrometry information. Additionally, we hope to utilize centrosome isolation and enrichment procedures to assist in identifying proteins which interact with Y130 dynein.

The Role of microRNAs in Retinal Regeneration in Zebrafish

Jessica Spiewak Coauthors: Rachel Harding Advisor: David Hyde, Dept. of Biological Sciences

Zebrafish (Danio rerio) have the natural ability to regenerate damaged photoreceptor cells in the retina. This ability is facilitated by Müller glia cells, which are able to reenter the cell cycle and proliferate within the inner nuclear layer of the retina following retinal damage. The Müller glia give rise to neuronal progenitor cells, which divide and migrate into the outer nuclear layer where they eventually become rods or cones. To better understand the mechanism behind this process, our lab focuses on genes involved in retinal regeneration. A previous microarray in our lab showed rapid and widespread changes in gene expression, which may be controlled by microRNAs. MiRNAs are endogenous short RNA sequences with the ability to regulate gene expression by binding and preventing translation of specific mRNA sequences. To examine the possible involvement of miRNAs in the retinal regeneration process, adult albino zebrafish were injected with a morpholino specific to the dicer mRNA, which encodes a protein necessary for the maturation of all miRNAs. Morpholino-mediated knockdown of Dicer protein expression was confirmed by Western blot, while immunohistochemistry analysis of proliferating Müller glia indicated that Dicer function is essential to an optimal proliferation response by the Müller glia following light damage. These results suggest miRNAs are participating in regulating the expression of genes involved in the proliferation response. Future experiments will focus on characterizing the role of miRNA-199 in the regeneration process. This miRNA has been shown to control vsx2 gene expression, which is responsible for proliferation of neuronal progenitor cells and bipolar cell identities in retinal development.

Development of New Techniques to Introduce Exogenous DNA into Adult Retinal Cells of Zebrafish for Protein Over-expression

Catherine Turner Advisor: David Hyde, Dept. of Biological Sciences

In order to further the investigations on neuronal regeneration in adult zebrafish (Danio rerio) retinas, a series of experiments tested new techniques to over-express exogenous proteins within adult retinal cells. To do this, the green florescence protein (GFP) genetic sequence under the control of the ubiquitous EF-1 alpha promoter was used for injection to easily track expression within the fish eye through fluorescence microscopy. Two carrier proteins were tested to evaluate their efficacy in delivering GFP into the cell body and directing transcription: the Escherichia coli single-stranded binging protein and a cationic “Peptide for Ocular Delivery” (POD). Both of these proteins use its net positive charge to mask the highly negative charge of the DNA and thus help to infiltrate the negatively charged cell membrane and rapidly introduce the new DNA construct, allowing it to be expressed. Experiments were performed using various concentrations of both the DNA and the carrier protein. GFP expression was examined with and without ocular electroporation. The use of the E. coli single-stranded binding protein failed to produce any florescence within the retinal cells. The POD peptide sequence, which was used successfully to introduce transgenes into rodent retinas, failed to produce GFP expression in the zebrafish eyes. These results suggest an inability of protein coating to successfully introduce and express exogenous DNA into the adult zebrafish retina.

Unique Thermal Hysteresis Antifreeze Glycolipid Confers Cryoprotection to Non-cold-tolerant Geranium Cells

Celeste Warrell Coauthor: Philip Nickell Advisor: John Duman, Dept. of Biological Sciences

Surviving subzero temperatures involves one of two strategies, freeze-avoidance or freeze- tolerance. Freeze-avoiding species cannot survive freezing, while freeze-tolerant species survive freezing in the extracellular space and actually promote ice formation in the extracellular fluid. However, cytoplasmic freezing is lethal, even in freeze-tolerant species. Biomolecules that contribute to the organism’s survival include antifreeze proteins, which bind to ice crystals, making continued ice growth impossible until the temperature is lowered. This ice-binding activity produces a difference between the ice crystal’s melting and freezing point, a phenomenon known as thermal hysteresis.Antifreeze proteins are usually associated with freeze-avoiding species, but many freeze-tolerant species exhibit thermal hysteresis as well. A recently discovered antifreeze glycolipid located on cell membranes and having antifreeze protein-like activity was isolated from the freeze-tolerant Alaskan beetle, Upis ceramboides. We hypothesize that this antifreeze glycolipid prevents the spread of extracellular ice into the cytoplasm by virtue of its position on the membrane and its ability to bind to ice, thereby preventing lethal cytoplasmic freezing. A similar and possibly identical antifreeze glycolipid has been identified in the freeze-tolerant plant Solanum dulcamara, a more easily-accessed source of the antifreeze glycolipid. The goal of this project was to determine whether the Solanum antifreeze glycolipid (AFGL) has a cryoprotective effect on non-cold tolerant geranium protoplasts (Pelargonium hortarum). After developing cell viability assays to establish baseline cell survivorship values under different parameters (varying temperatures, time of freezing, buffers, and vital dyes), an assay using fluorescein diacetate and epifluorescence microscopy was chosen. Geranium cells were frozen at subzero temperatures with and without the AFGL to compare cell survivorship between experimental and control sets. Preliminary evidence suggests that a highly diluted sample (1:20 dilution) of the Solanum AFGL having an original thermal hysteresis activity of ~2.4°C significantly enhances protoplast survivorship at −5oC, with a difference in percent survivorship of ~20%. Greater and lesser dilutions of this Solanum AFGL did not enhance protoplast survivorship at −5oC. Further research to determine the mechanism of the AFGL’s cryoprotection is currently underway.

Zw10 and SNX4 form the Basis for Dynein’s Vesicle Binding Complex

Michael Winding Advisor: Kevin Vaughan, Dept. of Biological Sciences

The dimeric motor protein, dynein, is involved in various essential cellular processes. Through its interaction with dynactin, dynein is tethered to kinetochore complexes in mitosis and to vesicles and organelles in interphase. Traveling along microtubules using its symmetric heavy chains, dynein drags its cargo in a negatively oriented direction toward the centrosomes. The activity of dynein and dynactin during mitosis has been extensively studied and characterized. By interacting with proteins such as zw10 and through differential phosphorylation, dynein has been shown to strip anaphase inhibitors from the kinetochore, allowing chromosomes to separate. On the other hand, Dynein’s role during interphase and the proteins that tether dynein to vesicles and organelles has been less explored. By investigating vesicle transport from the ER to the Golgi apparatus, the complex of proteins regulating dynein-vesicle attachment can be determined. Using a PI3Kinase inhibitor, ly294002, and a membrane fractionation protocol, various proteins have been categorized as possible candidates for creating a binding platform between dynein and vesicles. These proteins include the p150 subunit of dynactin, zw10, Rab6A, SNX1, and SNX4. Brefeldin-A washout of cos7 cells cotransfected with Nagt-GFP and zw10 siRNA compared with control cells revealed that inhibition of zw10 lowered the number of negatively directed vesicles as well as overall Golgi recovery. This indicates that zw10 helps form a connection between dynein and ER vesicles. Sorting nexin 4 (SNX4), a protein containing a BAR domain, has been shown to associate with membranes and has been implicated in intracellular trafficking (Haft et al, 1998). Sorting nexin 4 has also been shown to preferentially bind PI3P, a membrane lipid. Transfection of a SNX4 mutant, lacking a PI3P binding domain, caused decreased minus-oriented movement events as well as decreased movement distances. Brefeldin-A washout of cos7 cells transfected with a SNX4 mutant caused slower Golgi recovery, indicating that SNX4 is involved in ER-Golgi trafficking. Mass spectrometry of p150 IPs revealed a diverse population of proteins contained on ER-Golgi vesicles. These proteins and this study provide a roadmap for future research in the regulation of dynein-driven trafficking.