Inhibition of histone deacetylase 6 (HDAC6) was shown to support axon growth on the nonpermissive substrates myelin-associated glycoprotein (MAG) and chondroitin sulfate proteoglycans (CSPGs). Though HDAC6 deacetylates α-tubulin, we find that another HDAC6 substrate contributes to this axon growth failure. HDAC6 is known to impact transport of mitochondria, and we show that mitochondria accumulate in distal axons after HDAC6 inhibition. Miro and Milton proteins link mitochondria to motor proteins for axon transport. Exposing neurons to MAG and CSPGs decreases acetylation of Miro1 on Lysine 105 (K105) and decreases axonal mitochondrial transport. HDAC6 inhibition increases acetylated Miro1 in axons, and acetyl-mimetic Miro1 K105Q prevents CSPG-dependent decreases in mitochondrial transport and axon growth. MAG- and CSPG-dependent deacetylation of Miro1 requires RhoA/ROCK activation and downstream intracellular Ca 2+ increase, and Miro1 K105Q prevents the decrease in axonal mitochondria seen with activated RhoA and elevated Ca 2+ . These data point to HDAC6-dependent deacetylation of Miro1 as a mediator of axon growth inhibition through decreased mitochondrial transport.

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that β-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 µm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.

Lis1 and Ndel1 are essential for animal development. They interact directly with one another and with cytoplasmic dynein. The developing brain is especially sensitive to reduced Lis1 or Ndel1 levels, as both proteins influence spindle orientation, neural cell fate decisions, and neuronal migration. We report here that Lis1 and Ndel1 reduction in a mitotic cell line impairs prophase nuclear envelope (NE) invagination (PNEI). This dynein-dependent process facilitates NE breakdown (NEBD) and occurs before the establishment of the bipolar spindle. Ndel1 phosphorylation is important for this function, regulating binding to both Lis1 and dynein. Prophase cells in the ventricular zone (VZ) of embryonic day 13.5 Lis1 +/− mouse brains show reduced PNEI, and the ratio of prophase to prometaphase cells is increased, suggesting an NEBD delay. Moreover, prophase cells in the VZ contain elevated levels of Ndel1 phosphorylated at a key cdk5 site. Our data suggest that a delay in NEBD in the VZ could contribute to developmental defects associated with Lis1–Ndel1 disruption.

Here, we use a mouse model (DBA/2J) to readdress the location of insult(s) to retinal ganglion cells (RGCs) in glaucoma. We localize an early sign of axon damage to an astrocyte-rich region of the optic nerve just posterior to the retina, analogous to the lamina cribrosa. In this region, a network of astrocytes associates intimately with RGC axons. Using BAX-deficient DBA/2J mice, which retain all of their RGCs, we provide experimental evidence for an insult within or very close to the lamina in the optic nerve. We show that proximal axon segments attached to their cell bodies survive to the proximity of the lamina. In contrast, axon segments in the lamina and behind the eye degenerate. Finally, the Wld s allele, which is known to protect against insults to axons, strongly protects against DBA/2J glaucoma and preserves RGC activity as measured by pattern electroretinography. These experiments provide strong evidence for a local insult to axons in the optic nerve.

Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein–β-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous β-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.

Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III 1–7 ). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III 1 (FNΔIII 1 ), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III 9–10 to a position close to the NH 2 -terminal assembly domain, as well as a large deletion spanning repeats III 4–7 , had no effect on assembly. In contrast, two deletions that included repeat III 2 , ΔIII 1–2 and ΔIII 2–5 , caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III 2 but not III 1 contains an FN binding site. Thus, these results pinpoint repeat III 2 as an important module for FN–FN interactions during fibril growth.

The actin bundles in Drosophila bristles run the length of the bristle cell and are accordingly 65 microns (microchaetes) or 400 microns (macrochaetes) in length, depending on the bristle type. Shortly after completion of bristle elongation in pupae, the actin bundles break down as the bristle surface becomes chitinized. The bundles break down in a bizarre way; it is as if each bundle is sawed transversely into pieces that average 3 microns in length. Disassembly of the actin filaments proceeds at the "sawed" surfaces. In all cases, the cuts in adjacent bundles appear in transverse register. From these images, we suspected that each actin bundle is made up of a series of shorter bundles or modules that are attached end-to-end. With fluorescent phalloidin staining and serial thin sections, we show that the modular design is present in nondegenerating bundles. Decoration of the actin filaments in adjacent bundles in the same bristle with subfragment 1 of myosin reveals that the actin filaments in every module have the same polarity. To study how modules form developmentally, we sectioned newly formed and elongating bristles. At the bristle tip are numerous tiny clusters of 6-10 filaments. These clusters become connected together more basally to form filament bundles that are poorly organized, initially, but with time become maximally cross-linked. Additional filaments are then added to the periphery of these organized bundle modules. All these observations make us aware of a new mechanism for the formation and elongation of actin filament bundles, one in which short bundles are assembled and attached end-to-end to other short bundles, as are the vertical girders between the floors of a skyscraper.

The lamin B receptor (LBR) is a polytopic integral membrane protein localized exclusively in the inner nuclear membrane domain of the nuclear envelope. Its cDNA deduced primary structure consists of a highly charged amino-terminal domain of 205 residues that faces the nucleoplasm followed by a hydrophobic domain with eight potential transmembrane segments. To identify determinants that sort LBR from its site of integration (RER and outer nuclear membrane) to the inner nuclear membrane, we prepared full-length, truncated, and chimeric cDNA constructs of chick LBR, transfected these into mammalian cells and detected the expressed protein by immunofluorescence microscopy using appropriate antibodies. Surprisingly, we found that the determinants for sorting of LBR to the inner nuclear membrane reside in a region comprising its first transmembrane sequence plus flanking residues on either side. The other transmembrane regions as well as the nucleoplasmic domain are not required for sorting. We propose that the first transmembrane segment of LBR interacts specifically with another transmembrane segment and consider several mechanisms by which such specific interaction could result in sorting to the inner nuclear membrane.

To investigate the role of the intermediate filament protein vimentin in the normal differentiation and morphogenesis of the eye lens fiber cells, we generated transgenic mice bearing multiple copies of the chicken vimentin gene. In most cases, the vimentin transgene was overexpressed in the lenses of these animals, reaching up to 10 times the endogenous levels. This high expression of vimentin interfered very strongly with the normal differentiation of the lens fibers. The normal fiber cell denucleation and elongation processes were impaired and the animals developed pronounced cataracts, followed by extensive lens degeneration. The age of appearance and extent of these abnormalities in the different transgenic lines were directly related to the vimentin level. Electron microscopic analysis revealed that the accumulated transgenic protein forms normal intermediate filaments.

Pulmonary endothelial cells are capable of metabolizing a variety of circulating hormonal substances. Indirect evidence indicates that some of the relevant enzymes are located on the plasma membrane. The associated caveolae are of special interest as globular subunits, possibly enzyme clusters, are evident in their membranes. In the present study, freeze-etch techniques were used to improve understanding of the fine structure of endothelial cells and to extend our investigations of possible sites of enzymes capable of metabolizing circulating vasoactive agents. As in other cells studied by freeze-etching, intramembranous particles are found on both inner aspects of the plasma membrane. In undifferentiated areas of plasma membrane, the particles appear to have a random distribution. These areas fracture such that approximately equal proportions of the particles adhere to the cytoplasmic aspect of the outer leaflet and the extracellular aspect of the inner leaflet. However, the particles organize into rosettes and plaques at the base of caveolae, and, after fracture, the rosettes and plaques adhere predominantly to the cytoplasmic aspect of the outer leaflet. The peculiar organization of particles in association with caveolae supports the concept that caveolae have a stomal skeletal structure and raises the possibility that the organization may be in some way related to pinocytosis.

The fine structure of synapses in the central nervous system of lamprey ( Petromyzon marinus ) ammocoetes has been investigated. Both synapses within the neuropil and synaptic links between giant fibers (including Müller cells) and small postsynaptic units are described. The distribution of neurofilaments and microtubules in nerve profiles over a wide diameter range is described, and the possible role of these structures in intracellular transport is discussed. Electron micrographs indicate that small lucent "synaptic vesicles" occur sparsely throughout the axoplasm and in regular arrays in association with microtubules in the vicinity of synapses. Within a synaptic focus, immediately adjoining the presynaptic membrane, vesicles are randomly arranged and are not associated with microtubules. Neurofilaments are present, generally in large numbers, but these are not associated with vesicles or other particulates. The structural findings are considered in terms of current concepts of fast and slow transport in neurons and the mechanochemical control of intracellular movement of materials.

The organization of intersegmental muscle fibers associated with the dorsal abdominal sclerites of the cockroach is described. These fibers correspond closely, in the disposition and derivation of the membranes of the transverse tubular system and sarcoplasmic reticulum cisternae, with insect synchronous flight muscle fibers, but differ markedly from these in their fibrillar architecture and mitochondrial content. The mitochondria are small and generally aligned alongside the prominent I bands of the sarcomere, and, in the best-oriented profiles of the A bands, thick filaments are associated with orbitals of twelve thin filaments, a configuration that has also been observed in striated fibers of insect visceral muscle. These structural features of insect muscles are compared and discussed in terms of possible variations in the control of contraction and relaxation, and in the nature of their mechanical role.

The cytological organization of flight muscle fibers of Odonata has been investigated. These fibers, in representatives of the Zygoptera and Anisoptera, have been compared and found to be similar, except that, in the former, pairs of lamellar fibrils, rather than single fibrils, alternate with the mitochondria. In each instance, in these synchronous muscles, the actin filaments of the myofibrils are found to lie opposite to and midway between pairs of myosin filaments—a configuration previously reported in asynchronous flight muscle fibers. The disposition of the T system and sarcoplasmic reticulum membranes in glutaraldehyde-fixed anisopteran muscle is described in detail: the T system tubules are shown to be radially continuous across the fiber, and are derived as openmouthed invaginations from the surface cell-membrane. The detailed organization of the dyad junctions between these tubules and the adjoining cisternae of the sarcoplasmic reticulum is described. The accessibility of the T system interior to diffusion exchange with the general extracellular milieu has been investigated by studies on the penetration of ferritin into the fiber: molecules of this marker have been found to diffuse solely along the T system tubules, and their presence in the tubule extremities adjoining the centrally placed nuclei confirms the morphological evidence suggesting that these tubules provide open diffusion channels extending across the radius of the fiber. The possible physiological role of these membrane components and their distribution in synchronous muscles of insects and vertebrates and in asynchronous insect flight muscle are discussed.

The organization of the indirect flight muscle of an aphid (Hemiptera-Homoptera) is described. The fibers of this muscle contain an extensive though irregularly disposed complement of T system tubules, derived as open invaginations from the cell surface and from the plasma membrane sheaths accompanying the tracheoles within the fiber. The sarcoplasmic reticulum is reduced to small vesicles applied to the T system surfaces, the intermembrane gap being traversed by blocks of electron-opaque material resembling that of septate desmosomes. The form and distribution of the T system and sarcoplasmic reticulum membranes in flight muscles of representatives of the major insect orders is described, and the extreme reduction of the reticulum cisternae in all asynchronous fibers (to which group the aphid flight muscle probably belongs), and the high degree of their development in synchronous fibers is documented and discussed in terms of the contraction physiology of these muscle cells.

The distribution of esterase activity in the last abdominal ganglion, the connectives and the cereal nerves of the cockroach Periplaneta americana has been investigated cytochemically. Activity of an unspecific eserine-insensitive esterase (or esterases) has been found in glial elements in these regions of the nerve cord. In addition, sites of cholinesterase (eserine-sensitive) activity have been found in association with ( a ) the glial sheaths of the axons in the cereal nerves and connectives, ( b ) the glial folds encapsulating the neuron perikarya in the ganglion, and ( c ) in localized areas along the membranes of axon branches within the neuropile, often flanked by focal clusters of synaptic vesicles. These results are discussed with particular reference to the previously reported insensitivity of the insect nerve cord to applied acetylcholine, and to the probable existence of a cholinergic synaptic mechanism in the central nervous system of this insect.

The distribution of ATPase activity in the asynchronous flight muscles of Calliphora erythrocephala (Diptera) was studied at a fine structural level, using preparations of teased fibers, both unfixed and after brief fixation in hydroxyadipaldehyde, incubated in a medium for the histochemical demonstration of myosin or actomyosin ATPase. In relaxed fibrils, activity was found confined to the A bands and was absent from the H zones as well as from the Z and I band regions. At high magnification, deposits of final product, lead phosphate, appeared primarily related to the thick filaments, or to short lateral extensions from them. Evidence was gathered which indicated that this enzyme activity was that of a triphosphatase which did not act on dinucleoside or non-nucleoside substrates.

The electron microscopic structure of sectioned indirect flight muscle fibers of the blowfly Calliphora is described. Particular attention is paid to the organization of the sarcosomes (mitochondria) of this tissue, and this description is accompanied by an account of the appearance of these bodies in negatively stained preparations. In sectioned material, it has been shown that these sarcosomes are similar to other mitochondria in the disposition of the outer and inner limiting membranes, but that the cristae, confluent with the latter, are unusually regular, and form parallel plates, containing circular fenestrations forming cylindrical channels within the matrix. Negatively stained preparations of disrupted sarcosomes reveal that both the outer limiting membrane and the cristae membranes bear large numbers of small particles, similar in appearance to those described by Fernández-Morgán and others in various mitochondria. In Calliphora , these particles consist of a sub-spherical "head" and a cylindrical "stalk," and appear to be arranged on the mitochondrial membranes either randomly distributed, or collected into circular or elongated groups. Recent suggestions concerning the nature of these submitochondrial particles are discussed, and an attempt is made to correlate the aspects of organization of Calliphora sarcosomes, revealed by conventional sectioning of the "intact" structures, and by negative staining of sarcosomal derivatives.

The organization of the luminescent organ of an adult firefly has been studied with the electron microscope, and particular attention has been given to the disposition of nerve terminals within the organ. The cytological structure of the cells of the tracheal system, the peripheral and terminal axons, the photocytes and the cells of the dorsal ("reflecting") layer is described. Previous observations on the peripheral course of nerve branches alongside the tracheal trunks at the level of the dorsal layer and photocyte epithelium have been confirmed, and specialised nerve endings containing axoplasmic components structurally identical with "synaptic vesicles" and "neurosecretory droplets" have been identified, not in association with the surface of the photocytes, but lying between the apposed surfaces of two components of the tracheal epithelium: the tracheal end-cell and the tracheolar cell. These cytological findings are discussed in terms of available biochemical and physiological evidence concerning the mechanism of light emission in the firefly, especially with respect to the possible role of chemical "transmitter" action in triggering a response in a luminescent effector system.

The structure of the flight muscle of a dragonfly ( Aeshna sp.) has been studied with the light and electron microscopes, and the organization of this specialized tubular muscle is described. This tissue is characterized by the great development of the sarcosomes, which are slab-like and are arranged within the fiber opposite each sarcomere of the radially oriented lamellar myofibrils. A well developed and highly ordered sarcoplasmic reticulum is present, consisting of perforated curtain-like cisternae extending across the face of each fibril, together with tubular invaginations of the fiber plasma membrane situated within indentations in the sarcosomes and traversing the fibril surface midway between the Z and M levels. The structure of these fibers, and notably the organization of the reticulum, is compared with that of other types of muscle, and the possible role of the two components of the sarcoplasmic reticulum in the contraction physiology of the dragonfly muscle fiber is discussed.