ABSTRACT Cilia are cellular antennae that are essential for human development and physiology. A large number of genetic disorders linked to cilium dysfunction are associated with proteins that localize to the ciliary transition zone (TZ), a structure at the base of cilia that regulates trafficking in and out of the cilium. Despite substantial effort to identify TZ proteins and their roles in cilium assembly and function, processes underlying maturation of TZs are not well understood. Here, we report a role for the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in TZ maturation in the Drosophila melanogaster male germline. We show that reduction of cellular PIP 2 levels through ectopic expression of a phosphoinositide phosphatase or mutation of the type I phosphatidylinositol phosphate kinase Skittles induces formation of longer than normal TZs. These hyperelongated TZs exhibit functional defects, including loss of plasma membrane tethering. We also report that the onion rings ( onr ) allele of Drosophila E xo84 decouples TZ hyperelongation from loss of cilium–plasma membrane tethering. Our results reveal a requirement for PIP 2 in supporting ciliogenesis by promoting proper TZ maturation.

ABSTRACT This paper describes a novel type of nuclear structure – nuclear lipid islets (NLIs). They are of 40–100 nm with a lipidic interior, and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] molecules comprise a significant part of their surface. Most of NLIs have RNA at the periphery. Consistent with that, RNA is required for their integrity. The NLI periphery is associated with Pol II transcription machinery, including the largest Pol II subunit, transcription factors and NM1 (also known as NMI). The PtdIns(4,5)P 2 –NM1 interaction is important for Pol II transcription, since NM1 knockdown reduces the Pol II transcription level, and the overexpression of wild-type NM1 [but not NM1 mutated in the PtdIns(4,5)P 2 -binding site] rescues the transcription. Importantly, Pol II transcription is dependent on NLI integrity, because an enzymatic reduction of the PtdIns(4,5)P 2 level results in a decrease of the Pol II transcription level. Furthermore, about half of nascent transcripts localise to NLIs, and transcriptionally active transgene loci preferentially colocalise with NLIs. We hypothesize that NLIs serve as a structural platform that facilitates the formation of Pol II transcription factories, thus participating in the formation of nuclear architecture competent for transcription.

ABSTRACT Phosphoinositides are a collection of lipid messengers that regulate most subcellular processes. Amongst the seven phosphoinositide species, the roles for phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) P 2 ] at the plasma membrane, such as in endocytosis, exocytosis, actin polymerization and focal adhesion assembly, have been extensively studied. Recent studies have argued for the existence of PtdIns(4,5) P 2 at multiple intracellular compartments, including the nucleus, endosomes, lysosomes, autolysosomes, autophagic precursor membranes, ER, mitochondria and the Golgi complex. Although the generation, regulation and functions of PtdIns(4,5) P 2 are less well-defined in most other intracellular compartments, accumulating evidence demonstrates crucial roles for PtdIns(4,5) P 2 in endolysosomal trafficking, endosomal recycling, as well as autophagosomal pathways, which are the focus of this Commentary. We summarize and discuss how phosphatidylinositol phosphate kinases, PtdIns(4,5) P 2 and PtdIns(4,5) P 2 -effectors regulate these intracellular protein and membrane trafficking events.

ABSTRACT Plasma membrane Ca 2+ ATPases (PMCAs, also known as ATP2B1–ATP2B4) are known targets of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) P 2 ], but if and how they control the PtdIns(4,5) P 2 pool has not been considered. We demonstrate here that PMCAs protect PtdIns(4,5) P 2 in the plasma membrane from hydrolysis by phospholipase C (PLC). Comparison of active and inactive PMCAs indicates that the protection operates by two mechanisms; one requiring active PMCAs, the other not. It appears that the mechanism requiring activity is the removal of the Ca 2+ required for sustained PLC activity, whereas the mechanism not requiring activity is PtdIns(4,5) P 2 binding. We show that in PMCA overexpressing cells, PtdIns(4,5) P 2 binding can lead to less inositol 1,4,5-triphosphate (Ins P 3 ) and diminished Ca 2+ release from intracellular Ca 2+ pools. Inspection of a homology model of PMCA suggests that PMCAs have a conserved cluster of basic residues forming a ‘blue collar’ at the interface between the membrane core and the cytoplasmic domains. By molecular dynamics simulation, we found that the blue collar forms four binding pockets for the phosphorylated inositol head group of PtdIns(4,5) P 2 ; these pockets bind PtdIns(4,5) P 2 strongly and frequently. Our studies suggest that by having the ability to bind PtdIns(4,5) P 2 , PMCAs can control the accessibility of PtdIns(4,5) P 2 for PLC and other PtdIns(4,5) P 2 -mediated processes.

Summary Phosphatidylinositol phosphates are important regulators of processes such as the cytoskeleton organization, membrane trafficking and gene transcription, which are all crucial for polarized cell growth. In particular, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) P 2 ] has essential roles in polarized growth as well as in cellular responses to stress. In the yeast Saccharomyces cerevisiae , the sole phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) Mss4p is essential for generating plasma membrane PtdIns(4,5) P 2 . Here, we show that Mss4p is required for yeast invasive growth in low-nutrient conditions. We isolated specific mss4 mutants that were defective in cell elongation, induction of the Flo11p flocculin, adhesion and cell wall integrity. We show that mss4-f12 cells have reduced plasma membrane PtdIns(4,5) P 2 levels as well as a defect in its polarized distribution, yet Mss4-f12p is catalytically active in vitro . In addition, the Mss4-f12 protein was defective in localizing to the plasma membrane. Furthermore, addition of cAMP, but not an activated MAPKKK allele, partially restored the invasive growth defect of mss4-f12 cells. Taken together, our results indicate that plasma membrane PtdIns(4,5) P 2 is crucial for yeast invasive growth and suggest that this phospholipid functions upstream of the cAMP-dependent protein kinase A signaling pathway.

Summary The prototypical transient receptor potential (TRP) channel is the major light-sensitive, and Ca 2+ -permeable channel in the microvillar photoreceptors of Drosophila . TRP channels are activated following hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) P 2 ] by the key effector enzyme phospholipase C (PLC). Mutants lacking TRP channels undergo light-dependent retinal degeneration, as a consequence of the reduced Ca 2+ influx. It has been proposed that degeneration is caused by defects in the Ca 2+ -dependent visual pigment cycle, which result in accumulation of toxic phosphorylated metarhodopsin–arrestin complexes (M PP –Arr2). Here we show that two interventions, which prevent accumulation of M PP –Arr2, namely rearing under red light or eliminating the C-terminal rhodopsin phosphorylation sites, failed to rescue degeneration in trp mutants. Instead, degeneration in trp mutants reared under red light was rescued by mutation of PLC. Degeneration correlated closely with the light-induced depletion of PtdIns(4,5) P 2 that occurs in trp mutants due to failure of Ca 2+ -dependent inhibition of PLC. Severe retinal degeneration was also induced in the dark in otherwise wild-type flies by overexpression of a bacterial PtdIns P n phosphatase (SigD) to deplete PtdIns(4,5) P 2 . In degenerating trp photoreceptors, phosphorylated Moesin, a PtdIns(4,5) P 2 -regulated membrane–cytoskeleton linker essential for normal microvillar morphology, was found to delocalize from the rhabdomere and there was extensive microvillar actin depolymerisation. The results suggest that compromised light-induced Ca 2+ influx, due to loss of TRP channels, leads to PtdIns(4,5) P 2 depletion, resulting in dephosphorylation of Moesin, actin depolymerisation and disintegration of photoreceptor structure.

Summary HIV-1 transcriptional activator (Tat) enables viral transcription and is also actively released by infected cells. Extracellular Tat can enter uninfected cells and affect some cellular functions. Here, we examine the effects of Tat protein on the secretory activity of neuroendocrine cells. When added to the culture medium of chromaffin and PC12 cells, Tat was actively internalized and strongly impaired exocytosis as measured by carbon fiber amperometry and growth hormone release assay. Expression of Tat mutants that do not bind to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) P 2 ] did not affect secretion, and overexpression of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), the major PtdIns(4,5) P 2 synthesizing enzyme, significantly rescued the Tat-induced inhibition of neurosecretion. This suggests that the inhibition of exocytosis may be the consequence of PtdIns(4,5) P 2 sequestration. Accordingly, expression of Tat in PC12 cells interfered with the secretagogue-dependent recruitment of annexin A2 to the plasma membrane, a PtdIns(4,5) P 2 -binding protein that promotes the formation of lipid microdomains that are required for exocytosis. In addition Tat significantly prevented the reorganization of the actin cytoskeleton necessary for the movement of secretory vesicles towards plasma membrane fusion sites. Thus, the capacity of extracellular Tat to enter neuroendocrine cells and sequester plasma membrane PtdIns(4,5) P 2 perturbs several PtdIns(4,5) P 2 -dependent players of the exocytotic machinery, thereby affecting neurosecretion. We propose that Tat-induced inhibition of exocytosis is involved in the neuronal disorders associated with HIV-1 infection.

Summary During embryonic development and in metastatic cancers, cells detach from the epithelium and migrate with persistent directionality. Directional cell migration is also crucial for the regeneration and maintenance of the epithelium and impaired directional migration is linked to chronic inflammatory diseases. Despite its significance, the mechanisms controlling epithelial cell migration remain poorly understood. Villin is an epithelial-cell-specific actin modifying protein that regulates epithelial cell plasticity and motility. In motile cells villin is associated with the highly branched and the unbranched actin filaments of lamellipodia and filopodia, respectively. In this study we demonstrate for the first time that villin regulates directionally persistent epithelial cell migration. Functional characterization of wild-type and mutant villin proteins revealed that the ability of villin to self-associate and bundle actin as well as its direct interaction with phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) P 2 ] regulates villin-induced filopodial assembly and directional cell migration. Our findings suggest that convergence of different signaling cascades could spatially restrict villin activity to areas of high PtdIns(4,5) P 2 and F-actin concentration to assemble filopodia. Furthermore, our data reveal the ability of villin to undergo actin- and PtdIns(4,5) P 2 -induced self-association, which may be particularly suited to coalesce and reorganize actin bundles within the filopodia.

In N1E-115 cells, neurite retraction induced by neurite remodelling factors such as lysophosphatidic acid, sphingosine 1-phosphate and semaphorin 3A require the activity of phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks). PIP5Ks synthesise the phosphoinositide lipid second messenger phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5) P 2 ], and overexpression of active PIP5K is sufficient to induce neurite retraction in both N1E-115 cells and cerebellar granule neurones. However, how PIP5Ks are regulated or how they induce neurite retraction is not well defined. Here, we show that neurite retraction induced by PIP5Kβ is dependent on its interaction with the low molecular weight G protein Rac. We identified the interaction site between PIP5Kβ and Rac1 and generated a point mutant of PIP5Kβ that no longer interacts with endogenous Rac. Using this mutant, we show that Rac controls the plasma membrane localisation of PIP5Kβ and thereby the localised synthesis of PtdIns(4,5) P 2 required to induce neurite retraction. Mutation of this residue in other PIP5K isoforms also attenuates their ability to induce neurite retraction and to localise at the membrane. To clarify how increased levels of PtdIns(4,5) P 2 induce neurite retraction, we show that mutants of vinculin that are unable to interact with PtdIns(4,5) P 2 , attenuate PIP5K- and LPA-induced neurite retraction. Our findings support a role for PtdIns(4,5) P 2 synthesis in the regulation of vinculin localisation at focal complexes and ultimately in the regulation of neurite dynamics.

The ezrin, radixin and moesin (ERM) proteins regulate cell membrane architecture in several cellular contexts. Current models propose that ERM activation requires a PtdIns(4,5) P 2 -induced conformational change, followed by phosphorylation of a conserved threonine. However, how these inputs contribute in vivo to orchestrate ERM activation is poorly understood. We addressed this issue by evaluating the contribution of PtdIns(4,5) P 2 and phosphorylation to the regulation of moesin during Drosophila development. Unexpectedly, we found that a form of moesin that cannot be phosphorylated displayed significant activity and could substitute for the endogenous product during wing morphogenesis. By contrast, we also show that PtdIns(4,5) P 2 binding is essential for moesin recruitment to the membrane and for its subsequent phosphorylation. Our data indicate that PtdIns(4,5) P 2 acts as a dosing mechanism that locally regulates ERM membrane recruitment and activation, whereas cycles of phosphorylation and dephosphorylation further control their activity once they have reached the cell cortex.

Phosphoinositides regulate numerous processes in various subcellular compartments. Whereas many stimuli trigger changes in the plasma-membrane PtdIns(4,5) P 2 concentration, little is known about its precursor, PtdIns(4) P , in particular whether there are stimulus-induced alterations independent of those of PtdIns(4,5) P 2 . We investigated plasma-membrane PtdIns(4) P and PtdIns(4,5) P 2 dynamics in insulin-secreting MIN6 cells using fluorescent translocation biosensors and total internal reflection microscopy. Loss of PtdIns(4,5) P 2 induced by phospholipase C (PLC)-activating receptor agonists or stimulatory glucose concentrations was paralleled by increased PtdIns(4) P levels. In addition, glucose-stimulated cells regularly showed anti-synchronous oscillations of the two lipids. Whereas glucose-induced PtdIns(4) P elevation required voltage-gated Ca 2+ entry and was mimicked by membrane-depolarizing stimuli, the receptor-induced response was Ca 2+ independent, but sensitive to protein kinase C (PKC) inhibition and mimicked by phorbol ester stimulation. We conclude that glucose and PLC-activating receptor stimuli trigger Ca 2+ - and PKC-dependent changes in the plasma-membrane PtdIns(4) P concentration that are independent of the effects on PtdIns(4,5) P 2 . These findings indicate that enhanced formation of PtdIns(4) P , apart from ensuring efficient replenishment of the PtdIns(4,5) P 2 pool, might serve an independent signalling function by regulating the association of PtdIns(4) P -binding proteins with the plasma membrane.

Rab GTPases of the Arabidopsis Rab-E subclass are related to mammalian Rab8 and are implicated in membrane trafficking from the Golgi to the plasma membrane. Using a yeast two-hybrid assay, Arabidopsis phosphatidylinositol-4-phosphate 5-kinase 2 (PtdIns(4) P 5-kinase 2; also known as PIP5K2), was shown to interact with all five members of the Rab-E subclass but not with other Rab subclasses residing at the Golgi or trans -Golgi network. Interactions in yeast and in vitro were strongest with RAB-E1d[Q74L] and weakest with the RAB-E1d[S29N] suggesting that PIP5K2 interacts with the GTP-bound form. PIP5K2 exhibited kinase activity towards phosphatidylinositol phosphates with a free 5-hydroxyl group, consistent with PtdIns(4) P 5-kinase activity and this activity was stimulated by Rab binding. Rab-E proteins interacted with PIP5K2 via its membrane occupancy and recognition nexus (MORN) domain which is missing from animal and fungal PtdIns(4) P 5-kinases. In plant cells, GFP:PIP5K2 accumulated at the plasma membrane and caused YFP:RAB-E1d to relocate there from its usual position at the Golgi. GFP:PIP5K2 was rapidly turned over by proteasomal activity in planta, and overexpression of YFP:PIP5K2 caused pleiotropic growth abnormalities in transgenic Arabidopsis . We propose that plant cells exhibit a novel interaction in which PIP5K2 binds GTP-bound Rab-E proteins, which may stimulate temporally or spatially localized PtdIns(4,5) P 2 production at the plasma membrane.

It has long been known that phosphoinositides are present in cellular membranes, but only in the past four decades has our understanding of their importance for proper cell function advanced significantly. Key to determining the biological roles of phosphoinositides is understanding the enzymes involved in their metabolism. Although many such enzymes have now been identified, there is still much to learn about their cellular functions. Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) are a group of kinases that catalyse the production of phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5) P 2 ]. As well as being a substrate for the enzymes phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI3K), PtdIns(4,5) P 2 acts as a second messenger in its own right, influencing a variety of cellular processes. In this Commentary, we review how PIP5Ks are modulated to achieve regulated PtdIns(4,5) P 2 production, and discuss the role of these proteins in different cellular processes.

Basic knowledge as to the subcellular location and dynamics of PLCβ isozymes is scant. Here, we report on the subcellular location of GFP-PLCβ1a and the use of total internal reflection fluorescence (TIRF) microscopy to examine the dynamics of GFP-PLCβ1a at the plasma membrane upon stimulation of Gq-coupled receptors. Using this technique, we observed PLCβ1a dissociation from the plasma membrane upon addition of agonist. An increase in intracellular calcium and a decrease in PtdIns(4,5) P 2 both coincided with a translocation of PLCβ1a from the plasma membrane into the cytosol. In order to differentiate between calcium and PtdIns(4,5) P 2 , rapamycin-induced heterodimerization of FRB and FKBP12 fused to 5-phosphatase IV was used to instantaneously convert PtdIns(4,5) P 2 into PtdIns(4) P . Addition of rapamycin caused PLCβ1a to dissociate from the plasma membrane, indicating that removal of PtdIns(4,5) P 2 is sufficient to cause translocation of PLCβ1a from the plasma membrane. In conclusion, PLCβ1a localization is regulated by its own substrate.

Epsin contains a phospholipid-binding ENTH domain coupled to C-terminal domain motifs that bind coated pit proteins. We examined how these domains interact to influence epsin function and localization in Dictyostelium . Although not required for global clathrin function, epsin was essential for constructing oval spores during development. Within the epsin protein, we found that features important for essential function were distinct from features targeting epsin to clathrin-coated pits. On its own, the phospholipid-binding ENTH domain could rescue the epsin-null phenotype. Although necessary and sufficient for function, the isolated ENTH domain was not targeted within clathrin-coated pits. The C-terminal domain containing the coated-pit motif was also insufficient, highlighting a requirement for both domains for targeting to coated pits. Replacement of the ENTH domain by an alternative membrane-binding domain resulted in epsin that sequestered clathrin and AP2 and ablated clathrin function, supporting a modulatory role for the ENTH domain. Within the ENTH domain, residues important for PtdIns(4,5) P 2 binding were essential for both epsin localization and function, whereas residue T107 was essential for function but not coated pit localization. Our results support a model where the ENTH domain coordinates with the clathrin-binding C-terminal domain to allow a dynamic interaction of epsin with coated pits.

Differentiating neurons extend membrane protrusions that develop into growing neurites. The driving force for neurite outgrowth is the dynamic actin cytoskeleton, which is regulated by actin-binding proteins. In this study, we describe for the first time, the role of profilin I and its ligand interactions in neuritogenesis of PC12 cells. High-level overexpression of wild-type profilin I had an inhibitory effect on neurite outgrowth. Low levels of profilin I did not disturb this process, but these cells developed many more filopodia along the neurite shafts. Low-level overexpression of mutant forms of profilin I changed one or more aspects of PC12 differentiation. Expression of a profilin I mutant that is defective in actin binding (profilin I R74E ) decreased neurite length and strongly inhibited filopodia formation. Cells expressing mutants defective in binding proline-rich ligands (profilin I W3A and profilin I R136D ) differentiated faster, developed more and longer neurites and more branches. The profilin I R136D mutant, which is also defective in phosphatidylinositol 4,5-bisphosphate binding, enhanced neurite outgrowth even in the absence of NGF. Parental PC12 cells treated with the ROCK inhibitor Y27632, differentiate faster and display longer neurites and more branches. Similar effects were seen in cells expressing profilin I WT , profilin I W3A and profilin I R74E . By contrast, the profilin I R136D -expressing cells were insensitive to the ROCK inhibitor, suggesting that regulation of profilin I by phosphatidylinositol 4,5-bisphosphate metabolism is crucial for proper neurite outgrowth. Taken together, our data show the importance of the interaction of profilin I with actin, proline-rich proteins and phosphatidylinositol 4,5-bisphosphate in neuronal differentiation of PC12 cells.

Successful cleavage of animal cells requires co-ordinated regulation of the actomyosin contractile ring and cleavage furrow ingression. Data from a variety of systems implicate phosphoinositol lipids and calcium release as potential regulators of this fundamental process. Here we examine the requirement for various steps of the phosphatidylinositol (PtdIns) cycle in dividing crane fly ( Nephrotoma suturalis ) spermatocytes. PtdIns cycle inhibitors were added to living cells after cleavage furrows formed and began to ingress. Inhibitors known to block PtdIns recycling (lithium), PtdIns phosphorylation (wortmannin, LY294002) or phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) P 2 ] hydrolysis [U73122 (U7)] all stopped or slowed furrowing. The effect of these drugs on cytokinesis was quite rapid (within 0-4 minutes), so continuous metabolism of PtdIns appears to be required for continued cleavage furrow ingression. U7 caused cleavage furrow regression concomitant with depletion of F-actin from the contractile ring, whereas the other inhibitors caused neither regression nor depletion of F-actin. That U7 depletes furrow-associated actin seems counterintuitive, as inhibition of phospholipase C would be expected to increase cellular levels of PtdIns(4,5) P 2 and hence increase actin polymerization. Our confocal images suggest, however, that F-actin might accumulate at the poles of U7-treated cells, consistent with the idea that PtdIns(4,5) P 2 hydrolysis may be required for actin filaments formed at the poles to participate in contractile ring assembly at the furrow.

Ezrin/radixin/moesin (ERM) proteins crosslink actin filaments to plasma membranes and are involved in the organization of the cortical cytoskeleton,especially in the formation of microvilli. ERM proteins are reported to be activated as crosslinkers in a Rho-dependent manner and are stabilized when phosphorylated at their C-terminal threonine residue to create C-terminal threonine-phosphorylated ERM proteins (CPERMs). Using a CPERM-specific mAb, we have shown, in vivo, that treatment with C3 transferase (a Rho inactivator) or staurosporine (a protein kinase inhibitor) leads to the dephosphorylation of CPERMs, the translocation of ERM proteins from plasma membranes to the cytoplasm and microvillar breakdown. We further elucidated that ERM protein activation does not require C-terminal phosphorylation in A431 cells stimulated with epidermal growth factor. In certain types of kidney-derived cells such as MDCK cells, however, ERM proteins appear to be activated in the absence of Rho activation and remain active without C-terminal phosphorylation. Interestingly, microinjection of an aminoglycoside antibiotic, neomycin, which binds to polyphosphoinositides, such as phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5) P 2 ],affected the activation of ERM proteins regardless of cell type. These findings not only indicate the existence of a Rho-independent activation mechanism of ERM proteins but also suggest that both Rho-dependent and-independent activation of ERM proteins require a local elevation of PtdIns(4,5) P 2 concentration in vivo.

Neuronal calcium sensor-1 (NCS-1) and its non-mammalian homologue,frequenin, have been implicated in a spectrum of cellular processes, including regulation of stimulated exocytosis of synaptic vesicles and secretory granules (SGs) in neurons and neuroendocrine cells and regulation of phosphatidylinositol 4-kinase beta activity in yeast. However, apart from these intriguing putative functions, NCS-1 and frequenin are relatively poorly understood. Here, the distribution, dynamics and function of NCS-1 were studied using PC12 cells that stably express NCS-1-EYFP (NCS-1 fused to enhanced yellow fluorescent protein) or that stably overexpress NCS-1. Fluorescence and electron microscopies show that NCS-1-EYFP is absent from SGs but is present on small clear organelles, some of which are just below the plasma membrane. Total internal reflection fluorescence microscopy shows that NCS-1-EYFP is associated with synaptic-like microvesicles (SLMVs) in growth cones. Overexpression studies show that NCS-1 enhances exocytosis of synaptotagmin-labeled regulated secretory organelles (RSOs) under basal conditions and during stimulation by UTP. Significantly, these studies implicate NCS-1 in the enhancement of both basal and stimulated phosphoinositide-dependent exocytosis of RSOs in PC12 cells, and they show that NCS-1 is distributed strategically to interact with putative targets on the plasma membrane and on SLMVs. These studies also reveal that SLMVs undergo both fast directed motion and highly hindered diffusive motion in growth cones, suggesting that cytoskeletal constituents can both facilitate and hinder SLMV motion. These results also reveal interesting similarities and differences between transport organelles in differentiated neuroendocrine cells and neurons.