EXCITABLE MEMBRANE ULTRASTRUCTURE : I. Freeze Fracture of Crayfish Axons

Cross-sectioned and cross-fractured crayfish axons display regions in which axon and Schwann cell surface membranes are regularly curved and project into the axoplasm. At these regions (projections) the two membranes run precisely parallel, separated by a gap of 130–140 Å. Longitudinal fractures through the axons expose the inner fractured surface of either the internal (face A) or the external (face B) leaflet of axon and adjacent Schwann cell surface membranes. On both membranes the projections appear as elongated structures oriented with the long axis parallel to the long axis of the nerve fiber. On face A of the axon surface membrane they are seen as elongated indentations 0.5–1.2-µm long, 0.12–0.15-µm wide. The indentations contain parallel chains of globules. The chains repeat every 120–125 Å and are oriented obliquely in such a way that if one looks at the axon surface from the extracellular space, the axis of the chains is skewed counterclockwise to the long axis of the indentations by an acute angle (most often 55–60°). The globules repeat along the chain every 80–85 Å. Globules of adjacent chains are in register in such a way that the axis on which globules of neighboring chains are aligned forms an angle of 75–85° with the axis of the chains. The complex structure can be defined as a globular array with a rhomboidal unit cell of 80–85 x 120–125 Å. On face B of the axon surface membrane the complementary image of these structures is seen. The projections of the Schwann cell surface membrane also contain groupings of globules; however, these differ from those in the axonal projections in size, pattern of aggregation, and fracture properties. Several possible interpretations of the meaning of these membrane specializations could be proposed. They could be: (a) structures involved in the mechanism of excitation, (b) regions of presumed metabolic couplings, and (c) areas of cell-to-cell adhesion.     

THE STRUCTURAL ORGANIZATION OF THE SEPTATE AND GAP JUNCTIONS OF HYDRA

The septate junctions and gap junctions of Hydra were studied utilizing the extracellular tracers lanthanum hydroxide and ruthenium red. Analysis of the septate junction from four perspectives has shown that each septum consists of a single row of hexagons sharing common sides of 50–60 A. Each hexagon is folded into chair configuration. Two sets of projections emanate from the corners of the hexagons. One set (A projections) attaches the hexagons to the cell membranes at 80–100-A intervals, while the other set (V projections) joins some adjacent septa to each other. The septate junctions generally contain a few large interseptal spaces and a few septa which do not extend the full length of the junction. Basal to the septate junctions the cells in each layer are joined by numerous gap junctions. Gap junctions also join the muscular processes in each layer as well as those which connect the layers across the mesoglea. The gap junctions of Hydra are composed of rounded plaques 0.15–0.5 µ in diameter which contain 85-A hexagonally packed subunits. Each plaque is delimited from the surrounding intercellular space by a single 40-A band. Large numbers of these plaques are tightly packed, often lying about 20 A apart. This en plaque configuration of the gap junctions of Hydra contrasts with their sparser, more widely separated distribution in many vertebrate tissues. These studies conclude that the septate junction may possess some barrier properties and that both junctions are important in intercellular adhesion. On a morphological basis, the gap junction appears to be more suitable for intercellular coupling than the septate junction.     

Septate junctions in the gastrodermal epithelium of Phialidium: A fine structural study utilizing ruthenium red

The septate junctions of the gastrodermis of the hydromedusa, Phialidium gregarium, are composed of septa (80 A thick) which bridge the gap (130 A) between the outer leaflets of the plasma membranes of adjacent cells. The septa are parallel walls, presumably continuous around the cells, and en face show a periodicity of 110 A. Examination of material fixed in a ruthenium red-containing mixture shows that this dye penetrates the interseptal compartments and illucidates the finer structure of the septa. A model of an interpretation of the three-dimensional structure of the junction is presented and relevance of the results to current theories of cell communication is discussed.     

Design,synthesis and biological evaluation of 1-Aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as novel microtubule destabilizers

A series of 1-aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as microtubule destabilizers were designed, synthesised and evaluated for anticancer activity. Based on bioisosterism, we introduced the tetrazole moiety containing the hydrogen-bond acceptors as B-ring of XRP44X analogues. The key intermediates ethyl 1-aryl-1H-tetrazole-5-carboxylates 10 can be simply and efficiently prepared via a microwave-assisted continuous operation process. Among the compounds synthesised, compound 6–31 showed noteworthy potency against SGC-7901, A549 and HeLa cell lines. In mechanism studies, compound 6–31 inhibited tubulin polymerisation and disorganised microtubule in SGC-7901 cells by binding to tubulin. Moreover, compound 6–31 arrested SGC-7901cells in G2/M phase. This study provided a new perspective for development of antitumor agents that target tubulin.     

MORPHOLOGICAL CORRELATES OF INCREASED COUPLING RESISTANCE AT AN ELECTROTONIC SYNAPSE

Close appositions between axonal membranes are present in the septum between adjacent axonal segments of the septate or lateral giant axons of the crayfish Procambarus. In sections the closely apposed membranes appear separated by a space or gap. The use of lanthanum indicates that there may be structures connecting the apposed membranes. The apparent gap is actually a network of channels continuous with the extracellular space. Adjacent axonal segments are electrotonically coupled at the septa. The coupling resistance is increased by mechanical injury of an axon, immersion in low Cl^- solutions, and immersion in low Ca⁺⁺ solutions, followed by a return to normal physiological solution. Septa at which coupling resistance had been measured were examined in the electron microscope. The induced increases in coupling resistance are associated with separation of the junctional membranes (with the exception of the moderate increases during immersion in low Ca⁺⁺ solutions). Schwann cell processes are present between the separated axonal membranes. When nerve cords in low Cl^- solutions are returned to normal physiological solution, coupling, i.e., electrotonic synapses. A model of an electrotonic synapse is proposed in which tween axonal membranes are again found. The association between the morphological and physiological findings provides further evidence that the junctions are the sites of electrotonic coupling, i.e., electrotonic, synapses. A model of an electrotonic synapse is proposed in which intercytoplasmic channels not open to the extracellular space are interlaced with a hexagonal network of extracellular channels between the apposed junctional membranes.     

Microtubule-associated smooth endoplasmic reticulum in the frog's brain

Summary Electron microscopic studies of neural processes in the cerebellum, optic tectum, and cerebral hemisphere of the frog reveal a distinctive system of SER cisternae lying at intervals (commonly 1–2 m apart) perpendicular to the long axis of axons and dendrites, interconnected by tubular, longitudinally orientated SER elements, and in direct continuity with the outer membrane of mitochondria. The transverse cisternae are fenestrated, with a single mierotubule (or rarely, two) passing through the centre of each 50–75 nm fenestration. Extensions of the SER-microtubule complex may be located parasynaptically in axon terminals and dendrites. The SER of dendritic spines also appears to be continuous with the fenestrated cisternae.Possible roles for the specialized SER (particularly of the parasynaptic extensions), such as calcium ion sequestration and ATP or monoamine oxidase transport, are discussed.Thanks are due to Profs. E. G. Gray and J. Z. Young for helpful discussion and to Mrs. N. Morgan and Mr. R. Boddy for technical assistance.     

Fine structure of the autonomic nerves of the rabbit myometrium

Summary The fine structure of the preterminal nerve fibers of the rabbit myometrial smooth muscle was studied using potassium permanganate fixation or glutaraldehyde fixation with postosmification. The preterminal fibers were mostly formed by 2–10 axons enveloped by Schwann cells. Two kinds of axons and axon terminals were found. (1) Adrenergic axons, which contained many small, granular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å) which represented ca. 2% of the total count of the vesicles. (2) Nonadrenergic axons, which contained small agranular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å). Both types of axons formed preterminal varicosities along their course. The real terminal varicosities, representing the anatomical end of the axons, were usually larger than the preterminal ones and showed close contact to the plasma membranes of the smooth muscle cells. Both adrenergic and nonadrenergic terminals were found close to the smooth muscle cells, but a gap of at least 2000 Å was always present between the two cell membranes. The axons and preterminal varicosities of both types of nerves were in intimate contact with each other within the preterminal nerve fiber. Axo-axonal interactions between the two types of axons are possible in the rabbit myometrium. The relative proportion of the nonadrenergic axons from the total was about one fourth.     

Mutations in the β-tubulin binding site for peloruside A confer resistance by targeting a cleft significant in side chain binding

Peloruside A is a microtubule-stabilizing macrolide that binds to β-tubulin at a site distinct from the taxol site. The site was previously identified by H-D exchange mapping and molecular docking as a region close to the outer surface of the microtubule and confined in a cavity surrounded by a continuous loop of protein folded so as to center on Y340. We have isolated a series of peloruside A-resistant lines of the human ovarian carcinoma cell line A2780(1A9) to better characterize this binding site and the consequences of altering residues in it. Four resistant lines (Pel A-D) are described with single-base mutations in class I β-tubulin that result in the following substitutions: R306H, Y340S, N337D and A296S in various combinations. The mutations are localized to peptides previously identified by Hydrogen-Deuterium exchange mapping, and center on a cleft in which the drug side chain appears to dock. The Pel lines are 10–15-fold resistant to peloruside A and show cross resistance to laulimalide but not to any other microtubule stabilizers. They show no cross-sensitivity to any microtubule destabilizers, nor to two drugs with targets unrelated to microtubules. Peloruside A induces G₂/M arrest in the Pel cell lines at concentrations 10–15 times that required in the parental line. The cells show notable changes in morphology compared with the parental line.Key words: Apeloruside A, laulimalide, paclitaxel, drug resistance, mitotic arrest, binding site, β-tubulin     

LOW RESISTANCE JUNCTIONS IN CRAYFISH : II. Structural Details and Further Evidence for Intercellular Channels by Freeze-Fracture and Negative Staining

Low resistance junctions between axons of crayfish ganglia are studied by freeze-fracture and negative staining. In freeze-fracture, fracture planes that go through a junctional membrane expose two faces, both internal, called face A and face B. Face A belongs to the internal membrane leaflet and faces the gap. Face B belongs to the external membrane leaflet and faces the axoplasm. Face A displays pits, 60–100 Å in diameter, arranged in a hexagonal array with a unit cell of ~200 Å. An ~25 Å bump is frequently seen at the center of each pit. Some pits are occupied by a globule ~125 Å in diameter, which displays a central depression ~25 Å in size. Face B contains globules also arranged in a fairly regular hexagonal pattern. The center-to-center distance between adjacent globules is most frequently ~200 Å; however, occasionally certain globules are seen separated by a distance as short as ~125 Å. The top surface of the globules occasionally displays a starlike profile and seems to contain a central depression ~25 Å in diameter. In negatively stained preparations of membranes from the nerve cord, two types of membranes are seen containing a fairly regular pattern. In one, globules ~95 Å in diameter form a hexagonal close packing with a unit cell of ~95 Å. In the other, globules of the same size are organized in a larger hexagonal array with a unit cell of ~155 Å (swollen arrangement). Some of the globules forming the swollen arrangement are seen containing six subunits. The six subunits form a hexagon which is skewed with respect to the main rows of hexagons in such a way that the subunits lie on rows which make an angle of ~37° with the main rows.     

Disease mutations in desmoplakin inhibit Cx43 membrane targeting mediated by desmoplakin–EB1 interactions

Mechanisms by which microtubule plus ends interact with regions of cell–cell contact during tissue development and morphogenesis are not fully understood. We characterize a previously unreported interaction between the microtubule binding protein end-binding 1 (EB1) and the desmosomal protein desmoplakin (DP), and demonstrate that DP–EB1 interactions enable DP to modify microtubule organization and dynamics near sites of cell–cell contact. EB1 interacts with a region of the DP N terminus containing a hotspot for pathogenic mutations associated with arrhythmogenic cardiomyopathy (AC). We show that a subset of AC mutations, in addition to a mutation associated with skin fragility/woolly hair syndrome, impair gap junction localization and function by misregulating DP–EB1 interactions and altering microtubule dynamics. This work identifies a novel function for a desmosomal protein in regulating microtubules that affect membrane targeting of gap junction components, and elucidates a mechanism by which DP mutations may contribute to the development of cardiac and cutaneous diseases.     

A DISTINCTIVE CELL CONTACT IN THE RAT ADRENAL CORTEX

Extensive cell contacts which resemble septate junctions occur between cells in the three major zones of the rat adrenal cortex. Characteristically, they extend between small intercellular canaliculi and the periendothelial space, frequently interrupted by gap junctions and rarely by desmosomes. Zonulae occludentes have not been identified in the adrenal cortex. Along this distinctive cell contact, the cell membranes of apposing cells are separated by 210–300 a bisected by irregularly spaced 100–150-A extracellular particles which are often circular in profile. In lanthanum preparations, these particles appear to form a continuous chain throughout the intercellular space and are visualized as an alveolate structure in sections parallel to the plane of the cell membrane. The cell membrane in the area of septate-like contact does not differ from nonjunctional areas of the cell membrane in freeze-fracture replicas. The cell contact retains its integrity after cell dispersion and after the separation of cell membranes from disrupted cells. The intercellular particles also persist after brief extraction in lipid solvents. Besides adherence, possible functions of this adrenal contact include maintenance of the width of the extracellular space, the provision of channels between intercellular canaliculi and the bloodstream, and utilization as cation depots. Similar structures are also present between adrenal cortical cells of several other species and between interstitial cells of the testis. This type of cell contact may, in fact, be a typical feature of steroid-hormone-secreting tissues in vertebrates.     

Structural Aspects of Saltatory Particle Movement

A variety of cells possess particles which show movements statistically different from Brownian movements. They are characterized by discontinuous jumps of 2–30 µ at velocities of 0.5–5 µ/sec or more. A detailed analysis of these saltatory, jumplike movements makes it most likely that they are caused by transmission of force to the particles by a fiber system in the cell outside of the particle itself. Work with isolated droplets of cytoplasm from algae demonstrates a set of fibers involved in both cytoplasmic streaming and saltatory motion, suggesting that both phenomena are related to the same force-generating set of fibers. Analysis of a variety of systems in which streaming and/or saltatory movement occurs reveals two types of fiber systems spatially correlated with the movement, microtubules and 50 A microfilaments. The fibers in Nitella (alga) are of the microfilament type. In other systems (melanocyte processes, mitotic apparatus, nerve axons) microtubules occur. A suggestion is made, based on work on cilia, that a microtubule-microfilament complex may be present in those cases in which only microtubules appear to be present, with the microfilament closely associated with or buried in the microtubule wall. If so, then microfilaments, structurally similar to smooth muscle filaments, may be a force-generating element in a very wide variety of saltatory and streaming phenomena.     

Axonal Transport: How High Microtubule Density Can Compensate for?Boundary Effects in Small-Caliber Axons

Long-distance intracellular axonal transport is predominantly microtubule-based, and its impairment is linked to neurodegeneration. In this study, we present theoretical arguments that suggest that near the axon boundaries (walls), the effective viscosity can become large enough to impede cargo transport in small (but not large) caliber axons. Our theoretical analysis suggests that this opposition to motion increases rapidly as the cargo approaches the wall. We find that having parallel microtubules close enough together to enable a cargo to simultaneously engage motors on more than one microtubule dramatically enhances motor activity, and thus minimizes the effects of any opposition to transport. Even if microtubules are randomly placed in axons, we find that the higher density of microtubules found in small-caliber axons increases the probability of having parallel microtubules close enough that they can be used simultaneously by motors on a cargo. The boundary effect is not a factor in transport in large-caliber axons where the microtubule density is lower.     

The fission yeast transforming acidic coiled coil-related protein Mia1p/Alp7p is required for formation and maintenance of persistent microtubule-organizing centers at the nuclear envelope

Microtubule-organizing centers (MTOCs) concentrate microtubule nucleation, attachment and bundling factors and thus restrict formation of microtubule arrays in spatial and temporal manner. How MTOCs occur remains an exciting question in cell biology. Here, we show that the transforming acidic coiled coil–related protein Mia1p/Alp7p functions in emergence of large MTOCs in interphase fission yeast cells. We found that Mia1p was a microtubule-binding protein that preferentially localized to the minus ends of microtubules and was associated with the sites of microtubule attachment to the nuclear envelope. Cells lacking Mia1p exhibited less microtubule bundles. Microtubules could be nucleated and bundled but were frequently released from the nucleation sites in mia1Δ cells. Mia1p was required for stability of microtubule bundles and persistent use of nucleation sites both in interphase and postanaphase array dynamics. The γ-tubulin–rich material was not organized in large perinuclear or microtubule-associated structures in mia1Δ cells. Interestingly, absence of microtubules in dividing wild-type cells prevented appearance of large γ-tubulin–rich MTOC structures in daughters. When microtubule polymerization was allowed, MTOCs were efficiently assembled de novo. We propose a model where MTOC emergence is a self-organizing process requiring the continuous association of microtubules with nucleation sites.     

Viscoelasticity of Tau Proteins Leads to Strain Rate-Dependent Breaking of?Microtubules during Axonal Stretch Injury: Predictions from a Mathematical Model

The unique viscoelastic nature of axons is thought to underlie selective vulnerability to damage during traumatic brain injury. In particular, dynamic loading of axons has been shown to mechanically break microtubules at the time of injury. However, the mechanism of this rate-dependent response has remained elusive. Here, we present a microstructural model of the axonal cytoskeleton to quantitatively elucidate the interaction between microtubules and tau proteins under mechanical loading. Mirroring the axon ultrastructure, the microtubules were arranged in staggered arrays, cross-linked by tau proteins. We found that the viscoelastic behavior specifically of tau proteins leads to mechanical breaking of microtubules at high strain rates, whereas extension of tau allows for reversible sliding of microtubules without any damage at small strain rates. Based on the stiffness and viscosity of tau proteins inferred from single-molecule force spectroscopy studies, we predict the critical strain rate for microtubule breaking to be in the range 22–44 s⁻¹, in excellent agreement with recent experiments on dynamic loading of micropatterned neuronal cultures. We also identified a characteristic length scale for load transfer that depends on microstructural properties and have derived a phase diagram in the parameter space spanned by loading rate and microtubule length that demarcates those regions where axons can be loaded and unloaded reversibly and those where axons are injured due to breaking of the microtubules.     

POROUS SUBSTRUCTURE OF THE GLOMERULAR SLIT DIAPHRAGM IN THE RAT AND MOUSE

The highly ordered, isoporous substructure of the glomerular slit diaphragm was revealed in rat and mouse kidneys fixed by perfusion with tannic acid and glutaraldehyde. The slit diaphragm was similar in both animal species and appeared as a continuous junctional band, 300–450 Å wide, consistently present within all slits formed by the epithelial foot processes. The diaphragm exhibited a zipper-like substructure with alternating, periodic cross bridges extending from the podocyte plasma membranes to a central filament which ran parallel to and equidistant from the cell membranes. The dimensions and spacing of the cross bridges defined a uniform population of rectangular pores approximately 40 by 140 Å in cross section and 70 Å in length. The total area of the pores was calculated to be about 2–3% of the total surface area of the glomerular capillaries. Physiological data indicate that the glomerular filter functions as if it were an isoporous membrane which excludes proteins larger than serum albumin. The similarity between the dimensions of the pores in the slit diaphragm and estimates for the size and shape of serum albumin supports the conclusion from tracer experiments that the slit diaphragm may serve as the principal filtration barrier to plasma proteins in the kidney.     

ELECTRON MICROSCOPY OF ORAL CELLS IN VITRO : II. Subsurface and Intracytoplasmic Confronting Cisternae in Strain KB Cells

Two special areas involving membranous components in strain KB cells were studied by electron microscopy. The first area described is that of the subsurface regions of two apposing cells in which flattened cisternae (one cisternae in each subsurface region) with membranes spaced 110–230 A apart were found in a confrontation alignment. The long dimension of the profiles of these cisternae ranges from 0.5 to 2 µ. At these intercellular contact areas, each cisterna is closely applied to the adjacent plasma membrane; the intervening space is 60–100 A. We have named the cisternae in these roughly symmetrical areas of cell contact the subsurface confronting cisternae. Communications between these cisternae and those of the rough-surfaced endoplasmic reticulum also were observed. The second area described is that of the intracytoplasmic confronting cisternae. These cisternae were observed as oval or round images about 0.3–1.4 µ in diameter, each image being composed of a pair of concentrically arranged confronting cisternae with membranes spaced 200–400 A apart. The apposing membranes of the two confronting cisternae are electron opaque, smooth, and free of ribosomes, whereas the unapposed membranes are less dense, scalloped, and associated with ribosomes. The spacing between the two intracytoplasmic confronting cisternae is 70–110 A.     

CILIARY MEMBRANE DIFFERENTIATIONS IN TETRAHYMENA PYRIFORMIS : Tetrahymena Has Four Types of Cilia

We have examined thin sections and replicas of freeze-fractured cilia of Tetrahymena pyriformis. The ciliary necklace located at the base of all freeze-fractured oral and somatic cilia has been studied in thin sections. Since electron-dense linkers have been found to connect both microtubule doublets and triplets to the ciliary membrane at the level of the necklace, the linkers and the associated necklace seem to be related to the transition region between the doublets and triplets of a cilium. Plaque structures, consisting of small rectangular patches of particles located distal to the ciliary necklace, are found in strain GL, but are absent in other strains examined in this study. In freeze-cleaved material, additional structural differentiations are observed in the distal region of the ciliary membranes of somatic and oral cilia. Somatic cilia contain many randomly distributed particles within their membrane. Oral cilia can be divided into three categories on the basis of the morphology of their freeze-fractured membranes: (a) undifferentiated cilia with very few randomly distributed particles: (b) cilia with particles arranged in parallel longitudinal rows spaced at intervals of 810–1080 Å that are located on one side of the cilium; and (c) cilia with patches of particles arranged in short rows oriented obliquely to the main axis of the cilium. The latter particles, found on one side of the cilium, seem to serve as attachment sites for bristles 375–750 Å long and 100 Å wide which extend into the surrounding medium. The particles with bristles are located at the tips of cilia in the outermost membranelle and may be used to detect food particles and/or to modify currents in the oral region so that food particles are propelled more efficiently into the buccal cavity. Examination of thin-sectioned material indicates that the particles in oral cilia which form the longitudinal rows could be linked to microtubule doublets. Linkage between microtubule doublets and adjacent membrane areas on one side of the cilium could modify the form of ciliary beat by restricting the sliding of the microtubules. It is suggested that membrane-microtubule interactions may form the basis for the various forms of ciliary beat observed in different organisms.     

Identification of a Microtubule-associated Motor Protein Essential for Dendritic Differentiation

The quintessential feature of the dendritic microtubule array is its nonuniform pattern of polarity orientation. During the development of the dendrite, a population of plus end–distal microtubules first appears, and these microtubules are subsequently joined by a population of oppositely oriented microtubules. Studies from our laboratory indicate that the latter microtubules are intercalated within the microtubule array by their specific transport from the cell body of the neuron during a critical stage in development (Sharp, D.J., W. Yu, and P.W. Baas. 1995. J. Cell Biol. 130:93– 104). In addition, we have established that the mitotic motor protein termed CHO1/MKLP1 has the appropriate properties to transport microtubules in this manner (Sharp, D.J., R. Kuriyama, and P.W. Baas. 1996. J. Neurosci. 16:4370–4375). In the present study we have sought to determine whether CHO1/MKLP1 continues to be expressed in terminally postmitotic neurons and whether it is required for the establishment of the dendritic microtubule array. In situ hybridization analyses reveal that CHO1/MKLP1 is expressed in postmitotic cultured rat sympathetic and hippocampal neurons. Immunofluorescence analyses indicate that the motor is absent from axons but is enriched in developing dendrites, where it appears as discrete patches associated with the microtubule array. Treatment of the neurons with antisense oligonucleotides to CHO1/MKLP1 suppresses dendritic differentiation, presumably by inhibiting the establishment of their nonuniform microtubule polarity pattern. We conclude that CHO1/MKLP1 transports microtubules from the cell body into the developing dendrite with their minus ends leading, thereby establishing the nonuniform microtubule polarity pattern of the dendrite.     

A Highlights from MBoC Selection: γ-Tubulin controls neuronal microtubule polarity independently of Golgi outposts

Neurons have highly polarized arrangements of microtubules, but it is incompletely understood how microtubule polarity is controlled in either axons or dendrites. To explore whether microtubule nucleation by γ-tubulin might contribute to polarity, we analyzed neuronal microtubules in Drosophila containing gain- or loss-of-function alleles of γ-tubulin. Both increased and decreased activity of γ-tubulin, the core microtubule nucleation protein, altered microtubule polarity in axons and dendrites, suggesting a close link between regulation of nucleation and polarity. To test whether nucleation might locally regulate polarity in axons and dendrites, we examined the distribution of γ-tubulin. Consistent with local nucleation, tagged and endogenous γ-tubulins were found in specific positions in dendrites and axons. Because the Golgi complex can house nucleation sites, we explored whether microtubule nucleation might occur at dendritic Golgi outposts. However, distinct Golgi outposts were not present in all dendrites that required regulated nucleation for polarity. Moreover, when we dragged the Golgi out of dendrites with an activated kinesin, γ-tubulin remained in dendrites. We conclude that regulated microtubule nucleation controls neuronal microtubule polarity but that the Golgi complex is not directly involved in housing nucleation sites.