Video-enhanced Differential Interference Contrast Research Articles

Actin cores of hair-cell stereocilia support myosin motility.

The actin cores of hair-cell stereocilia were tested as a substrate for the movement of myosin-coated beads in an in vitro assay. Large numbers of stereocilia from bullfrog sacculi and semicircular canals were isolated by blotting onto coverglasses and were demembranated to expose the polar actin tracks of their cytoskeletal cores. Silica or polystyrene beads, coated with thick filaments of chicken skeletal muscle myosin, were added to this core preparation in the presence of ATP. Myosin-coated beads could reach some of the cores by diffusion alone, but the efficiency and precision of the assay were improved considerably by the use of "optical tweezers" (a gradient-force optical trap) to deposit the beads directly on the cores. Beads applied in this fashion bound and moved unidirectionally at 1-2 microns/s, escaping the retarding force of the trap. Actin filaments within the stereocilia are cross-linked by fimbrin, but this did not appear to interfere with the motility of myosin. Beads coated with optic-lobe kinesin were also tested for movement; these bound and moved unidirectionally at 0.1-0.2 microns/s when applied to microtubule-based kinociliary cores, but not when applied to actin-based stereociliary cores. Our results are consistent with, and lend support to, a model for hair cell adaptation in which a molecular motor such as myosin maintains tension on the mechanically gated transduction channels. Optical tweezers and video-enhanced differential interference contrast optics provide high efficiency and improved optical resolution for the in vitro analysis of myosin motility.

Photoreceptor mosaic: Number and distribution of rods and cones in the rhesus monkey retina

Video-enhanced differential interference contrast optics was used to determine the number and distribution of photoreceptors across the entire retinal surface of 9 eyes obtained from 7 adult rhesus monkeys. We found that the retina of this primate contains an average of 3,100,000 cones (+/- 130,000) and 61,000,000 rods (+/- 7,500,000). Variation among animals in the number of rods and cones cannot be accounted for by differences in sex, age, or retinal surface area, nor is there a correlation between the number of rods and cones (a retina with a high number of rods does not typically have a high number of cones). Cone density peaks at 141,000 cones/mm2 in the foveola and decreases about 100-fold toward the periphery. Rod density in a central annulus around the fovea is 130,000/mm2 and decreases 6-8-fold toward the periphery. In all 9 retinae, we found that an area 4-5 mm dorsal to the fovea had the highest rod density at 184,000 rods/mm2. The functional significance of this area, which we term the dorsal rod peak (DRP), may be related to high sensitivity vision under scotopic conditions. Outside of the DRP, rod density is symmetrical around the major axes of the retina, whereas cone density is elevated in nasal retina. Among animals, both rods and cones display a 2-fold individual difference in receptor density at any given eccentricity. Although rods and cones differ in absolute number, the location and magnitude of their peak densities, and their central to peripheral density gradients, the ratio of the density of rods to cones (15-30:1) is remarkably stable from 3 mm to 15 mm eccentricity. The relative consistency in the proportion of rods and cones in extrafoveal retina may be related to mechanisms of retinal development and functional interactions between scotopic and photopic systems.

Nucleation and growth of fibres and gel formation in sickle cell haemoglobin

Deoxygenated sickle haemoglobin polymerizes into long 210-A diameter fibres that distort and decrease the deformability of red blood cells, and cause sickle cell disease. The fibres consist of seven intertwined double strands. They can form birefringent nematic liquid crystals (tactoids) and spherulites. Rheologically, the system behaves as a gel. The equilibria show a phase separation and a solubility. The reaction kinetics show a delay time, are then roughly exponential and are highly dependent on concentration and temperature, and accord with the double nucleation model. But these conclusions are derived from macroscopic data, without direct observation of individual fibres. We have now used non-invasive video-enhanced differential interference contrast (DIC) and dark-field microscopy to observe nucleation, growth and interaction of sickle deoxyhaemoglobin fibres in real time. The fibres originate both from centres that produce many radially distributed fibres and on the surface of pre-existing fibres, from which they then branch. The resulting network is cross-linked and dynamic in that it is flexible and continues to grow and cross-link. Our results support most aspects of the double nucleation model.

Concentration of membrane antigens by forward transport and trapping in neuronal growth cones

Formation of the nervous system requires that neuronal growth cones follow specific paths and then stop at recognition signals, sensed at the growth cone's leading edge. We used antibody-coated gold particles viewed by video-enhanced differential interference contrast microscopy to observe the distribution and movement of two cell surface molecules, N-CAM and the 2A1 antigen, on growth cones of cultured cortical neurons. Gold particles are occassionally transported forward at 1–2 μm/s to the leading edge where they are trapped but continue to move. Concentration at the edge persists after cytochalasin D treatment or ATP depletion, but active movements to and along edges cease. We also observed a novel outward movement of small cytoplasmic aggregates at 1.8 μm/s in filopodia. We suggest that active forward transport and trapping involve reversible attachment of antigens to and transport along cytoskeletal elements localized to edges of growth cones.

Formation, transport, contraction, and disassembly of stress fibers in fibroblasts

Swiss mouse 3T3 fibroblasts grown on a solid substrate in the presence of 10% serum exhibit cell movement, organelle transport, and cytokinesis. When the serum concentration in the culture medium is decreased to 0.2% for 48 h the serum-deprived cells virtually stop locomoting, spread, decreased organelle transport, and exhibit extensive arrays of stress fibers that are visible with video-enhanced differential interference contrast microscopy and that also incorporate fluorescent analogs of actin and conventional myosin (myosin II). The stress fibers form in a constitutive manner at the cytoplasm-membrane interface, transport toward the nucleus, and then disappear. The rate of transport of these fibers is quite heterogeneous with average rates in the range of 10-20 microns/h. When serum-deprived cells are stimulated with mitogens such as 10% serum or 10 nM thrombin, many of the stress fibers immediately begin to shorten, suggesting a contraction. The rate of shortening is approximately two orders of magnitude slower than that of unloaded smooth muscle cells. The fiber shortening is often accompanied by retraction of the edges of the cell and continues for at least the 1st hour post-stimulation.

Dynamics of the endoplasmic reticulum and other membranous organelles in growth cones of cultured neurons

The fluorescent lipophilic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] was used to examine the distribution of membrane-bound organelles in growth cones of cultured rat sympathetic neurons. Within chemically fixed growth cones, intense DiOC6(3) fluorescence was localized predominately to the base or central region of growth cones. However, in most growth cones several thin DiOC6(3)-fluorescent processes radiated from the base into the periphery, and double fluorescence imaging of single growth cones indicated that these processes were highly colocalized (approximately 79%) with microtubules. The distribution of DiOC6(3) fluorescence in living growth cones was examined using low light-level fluorescence video microscopy. We observed thin fluorescent processes within the periphery of growth cones to undergo length excursions (extension/retraction) and to change orientation (move laterally). During growth cone advance, processes became progressively thicker and were gradually engulfed by the advancing fluorescent mass. When growth cones were viewed with video-enhanced differential interference contrast microscopy, the position of the fluorescent processes correlated with thickened extensions of central-type cytoplasm through which vesiclelike organelle transport often occurred. These observations indicate several features concerning the organization and movement of membranous organelles (MOs) in growth cones: (1) MOs are highly compartmentalized, the majority being localized to the growth cone base; (2) MOs advance into the periphery along distinct pathways probably associated with microtubules; (3) one or more thin continuous MOs, which most likely represent a thin tubular component of the endoplasmic reticulum, generally precedes advance of vesiclelike MOs along individual transport pathways; and (4) transport pathways with their associated MOs are spatially and temporally dynamic.

Elongated microvilli support the sea urchin embryo concentrically within the perivitelline space until hatching.

The early sea urchin embryo is supported in a concentric position within the perivitelline space by elongated microvilli which are attached to the fertilization envelope by extracellular matrix fibers. This "attachment complex," of microvillus tip: extracellular matrix fibers: fertilization envelope, was revealed by two methods: the use of pronase or calcium-free sea water to dissolve the extracellular matrix fibers, thus causing the eggs to lose their concentric location, and the visualization of the "attachment complex" using video-enhanced differential interference contrast microscopy and transmission electron microscope images. The presence of the "attachment complex" helps in understanding two types of early developmental events: (1) the apparently continual change in microvillus length during cleavage stages which retains the embryos in their concentric position and (2) the hatching process.

Three-dimensional counting: an accurate and direct method to estimate numbers of cells in sectioned material.

We introduce a way to count and measure cells in an optically defined volume of tissue called a counting box. This method--direct three-dimensional counting (3DC)--eliminates the need for correction factors, such as that introduced by Abercrombie (Anat. Rec. 94:239-247,'46), to determine the number of cells per unit volume (NV). Problems caused by irregular cell shape and cell size, nonrandom orientation, and splitting of cells by the knife during sectioning are overcome. Furthermore, 3DC is insensitive to large variations in section thickness. The innovative feature of 3DC is the definition of a counting box with top and bottom sides located inside the section a precise distance away from the cut surfaces of the tissue. The positions of the top and bottom sides of the counting box are delimited by using a digital length gauge and a Z-axis control unit. Sections of tissue between 8 and 100 micron thick are examined with a high numerical aperture objective in combination with video-enhanced differential interference contrast optics (DIC). Cells are marked on a television screen while the microscopist scans systematically from the top to the bottom of the counting box. Cells that are located completely inside the box and cells that only cross through its top, right, or back sides are counted. All cells that cross the planes that define the bottom, left, and front sides of the counting box are not counted. Direct 3DC provides an accurate, simple, and reliable way to count cells, nuclei, nucleoli, or other objects in sectioned material.(ABSTRACT TRUNCATED AT 250 WORDS)

Real-time observations of microtubule dynamic instability in living cells.

Individual microtubule dynamics were observed in real time in primary cultures of newt lung epithelium using video-enhanced differential interference contrast microscopy and digital image processing. The linear filaments observed in cells corresponded to microtubules based on three criteria: (a) small particles translocated along them; (b) the majority of them disappeared after incubation in nocodazole; (c) and the distribution observed by differential interference contrast correlated with anti-tubulin immunofluorescence staining of the same cell. Microtubules were most clearly observed at the leading edge of cells located at the periphery of the epithelial sheet. Microtubules exhibited dynamic instability behavior: individual microtubules existed in persistent phases of elongation or rapid shortening. Microtubules elongated at a velocity of 7.2 micron/min +/- 0.3 SEM (n = 42) and rapidly shortened at a velocity of 17.3 micron/min +/- 0.7 SEM (n = 35). The transitions between elongation and rapid shortening occurred abruptly and stochastically with a transition frequency of 0.014 s-1 for catastrophe and 0.044 s-1 for rescue. Approximately 70% of the rapidly shortening microtubules were rescued and resumed elongation within the 35 x 35 micron microscopic field. A portion of the microtubule population appeared differentially stable and did not display any measurable elongation or shortening during 10-15-min observations.

Micropruning: the mechanism of turning of Aplysia growth cones at substrate borders in vitro

Growth cones of Aplysia californica neurons were observed with video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy as they turned at a border between poly-L-lysine-treated and untreated glass. Growth cones that turned generally developed 2 distinct active areas of filopodial and veil formation, much in the way of growth cones undergoing branching. Both active areas advanced, but turning of the neurite occurred through the selective resorption of the incipient branches developing on the untreated substrate. Thus, micropruning of developing regions of the growth cone, rather than the asymmetric extension of filopodia or veils, was primarily responsible for directing neurite growth. We present the hypothesis that abrupt turns by growing neurites are mediated by 2 sets of signals, one causing growth cone splitting, and a second set regulating the survival of the separate branches.

The microtubule-dependent formation of a tubulovesicular network with characteristics of the ER from cultured cell extracts

The formation of a dynamic tubulovesicular membrane network that resembles the endoplasmic reticulum (ER) has been observed in extracts of cultured chick embryo fibroblasts (CEF cells) using video-enhanced differential interference contrast microscopy. Initially, membranes in the CEF extracts appeared amorphous and aggregated, but with time, membrane tubules moved out along stationary microtubules. The membrane tubules formed new branches on intersecting microtubules and fused with other branches to form a network of interconnected polygons. The tubulovesicular network was solubilized by detergent and took on a beaded morphology in a hypotonic buffer. Formation of the tubulovesicular network required ATP and microtubules. The network did not contain elements of the plasma membrane, Golgi apparatus, or mitochondria but could be labeled with ER markers. We suggest that the tubulovesicular network contains components from the ER and is formed by membrane associated motors moving upon microtubules in a process we call microtubule-dependent tethering.

Elimination of neurons from the rhesus monkey's lateral geniculate nucleus during development.

The timing, magnitude, and spatial distribution of neuron elimination was studied in the dorsal lateral geniculate nucleus of 57 rhesus monkeys (Macaca mulatta) ranging in age from the 48th day of gestation to maturity. Normal and degenerating cells were counted in Nissl-stained sections by using video-enhanced differential interference contrast optics and video-overlay microscopy. Before embryonic day 60 (E60), the geniculate nucleus contains 2,200,000 +/- 100,000 neurons. Roughly 800,000 of these neurons are eliminated over a 40- to 50-day period spanning the middle third of gestation. Neurons are lost at an average rate of 300 an hour between E48 and E60, and at an average rate of 800 an hour between E60 and E100. Very few neurons are lost after E100, and as early as E103 the population has fallen to the adult average of 1,400,000 +/- 90,000. Degenerating neurons are far more common in the magnocellular part of the nucleus than in the parvicellular part. In 20 of 29 cases, the number of neurons is greater on the right than on the left side. The right-left asymmetry averages about 8.5% and the difference is statistically significant (phi 2 = 38, p less than .001). The period of cell death occurs before the emergence of cell layers in the geniculate nucleus, before the establishment of geniculocortical connections, and before the formation of ocular dominance columns (Rakic, '76). Most important, the depletion of neurons in the geniculate nucleus begins long before the depletion of retinal axons. The number of geniculate neurons is probably a key factor controlling the number of the retinal cells that survive to maturity.

D1-type dopamine receptors inhibit growth cone motility in cultured retina neurons: evidence that neurotransmitters act as morphogenic growth regulators in the developing central nervous system.

Precedent exists for the early development and subsequent down-regulation of neurotransmitter receptor systems in the vertebrate central nervous system, but the function of such embryonic receptors has not been established. Here we show that stimulation of early-developing dopamine receptors in avian retina cells greatly inhibits the motility of neuronal growth cones. Neurons from embryonic chicken retinas were cultured in low-density monolayers, and their growth cones were observed with phase-contrast or video-enhanced-contrast-differential-interference-contrast (VEC-DIC) microscopy. Approximately 25% of the neurons responded to micromolar dopamine with a rapid reduction in filopodial activity followed by a flattening of growth cones and retraction of neurites. The response occurred at all ages examined (embryonic day-8 retinal neurons cultured on polylysine-coated coverslips for 1-7 days), although neurite retraction was greatest in younger cultures. Effects of dopamine on growth cone function could be reversed by haloperidol or (+)-SCH 23390, whereas forskolin elicited a response similar to dopamine; these data show the response was receptor-mediated, acting through a D1-type system, and are consistent with the use of cAMP as a second messenger. The experiments provide strong support for the hypothesis that neurotransmitters, besides mediating transynaptic signaling in the adult, may have a role in neuronal differentiation as growth regulators.

D1-type dopamine receptors inhibit growth cone motility in cultured retina neurons: evidence that neurotransmitters act as morphogenic growth regulators in the developing central nervous system.

Precedent exists for the early development and subsequent down-regulation of neurotransmitter receptor systems in the vertebrate central nervous system, but the function of such embryonic receptors has not been established. Here we show that stimulation of early-developing dopamine receptors in avian retina cells greatly inhibits the motility of neuronal growth cones. Neurons from embryonic chicken retinas were cultured in low-density monolayers, and their growth cones were observed with phase-contrast or video-enhanced-contrast-differential-interference-contrast (VEC-DIC) microscopy. Approximately 25% of the neurons responded to micromolar dopamine with a rapid reduction in filopodial activity followed by a flattening of growth cones and retraction of neurites. The response occurred at all ages examined (embryonic day-8 retinal neurons cultured on polylysine-coated coverslips for 1-7 days), although neurite retraction was greatest in younger cultures. Effects of dopamine on growth cone function could be reversed by haloperidol or (+)-SCH 23390, whereas forskolin elicited a response similar to dopamine; these data show the response was receptor-mediated, acting through a D1-type system, and are consistent with the use of cAMP as a second messenger. The experiments provide strong support for the hypothesis that neurotransmitters, besides mediating transynaptic signaling in the adult, may have a role in neuronal differentiation as growth regulators.

D1-type dopamine receptors inhibit growth cone motility in cultured retina neurons: evidence that neurotransmitters act as morphogenic growth regulators in the developing central nervous system.

Precedent exists for the early development and subsequent down-regulation of neurotransmitter receptor systems in the vertebrate central nervous system, but the function of such embryonic receptors has not been established. Here we show that stimulation of early-developing dopamine receptors in avian retina cells greatly inhibits the motility of neuronal growth cones. Neurons from embryonic chicken retinas were cultured in low-density monolayers, and their growth cones were observed with phase-contrast or video-enhanced-contrast-differential-interference-contrast (VEC-DIC) microscopy. Approximately 25% of the neurons responded to micromolar dopamine with a rapid reduction in filopodial activity followed by a flattening of growth cones and retraction of neurites. The response occurred at all ages examined (embryonic day-8 retinal neurons cultured on polylysine-coated coverslips for 1-7 days), although neurite retraction was greatest in younger cultures. Effects of dopamine on growth cone function could be reversed by haloperidol or (+)-SCH 23390, whereas forskolin elicited a response similar to dopamine; these data show the response was receptor-mediated, acting through a D1-type system, and are consistent with the use of cAMP as a second messenger. The experiments provide strong support for the hypothesis that neurotransmitters, besides mediating transynaptic signaling in the adult, may have a role in neuronal differentiation as growth regulators.

Optical approaches to the study of foraminiferan motility.

Microtubules are the major cytoskeletal component of foraminiferan reticulopodia. Video-enhanced differential interference contrast light microscopy has demonstrated that the microtubules serve as the intracellular tracks along which rapid bidirectional organelle transport and cell surface motility occurs. Microtubules appear to move, both axially and laterally within the pseudopodial cytoplasm, and these microtubule translocations appear to drive the various reticulopodial movements. F-actin is localized to discrete filament plaques form at sites of pseudopod-substrate adhesion. Correlative immunofluorescence and electron microscopy reveals a structural interaction between microtubules and the actin-containing filament plaques. Our recent data on reticulopodial motility are discussed in an historical context, and a model for foram motility, based on motile microtubules, is presented.

The translocation of mitochondria along insect ovarian microtubules from isolated nutritive tubes: a simple reactivated model.

Nutritive tubes, the microtubule-based translocation channels that link the trophic tissue to the developing oocytes in the ovaries of hemipteran insects, have been isolated and examined using video-enhanced differential interference contrast microscopy. When viewed in this way the nutritive tubes are seen to fray into linear strands, which, on the addition of exogenous ATP, support the translocation of particles along their lengths. The movement is also seen with GTP but not AMP-PNP. It is not affected by the addition of inhibitors of dynein or of energy metabolism. Electron microscopy shows the strands to consist of bundles of parallel microtubules of different sizes and the moving particles to be mitochondria. Comparisons are drawn between the movement of mitochondria along isolated insect ovarian microtubules and the reported translocation of vesicles along microtubules from squid axoplasm. The simplicity of the insect system is emphasized. The fact that it can be isolated easily and characterized biochemically makes it potentially valuable for investigating microtubule-based translocation.

Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons

This report examines cAMP-induced regulation of directed organelle transport in bag cell neuron growth cones using video-enhanced differential interference contrast (DIC) microscopy (Allen et al., 1981; Inoue, 1981) and digital image analysis techniques. Under control conditions, organelle transport is evident in the central cytoplasmic regions of bag cell neuron growth cones, but not in lamellae. Motility of lamellae takes the form of slow (less than 0.01 micron/sec) extension of margins and ruffling motions that propagate as waves (velocity, approximately 0.07 micron/sec) in a retrograde direction. Application of forskolin and a phosphodiesterase (PDE) inhibitor at concentrations known to induce changes in bag cell protein phosphorylation resulted in (1) rapid extension of directed organelle transport into lamellae, and (2) inhibition of the retrograde ruffling waves. These changes effected transformation of lamellae into neurite endings packed with microtubules and organelles, a large proportion of which appeared to be neurosecretory granules. The effects were reversible, dose-dependent, potentiated by a variety of PDE inhibitors, and mimicked by 6-N-butyl-8-benzyl-thio-cAMP (BT-cAMP). Though forskolin may normally promote depolarization and Ca entry, these changes in growth cone structure are not secondary to influx of external Ca, as they persist in Ca-free/EGTA solutions; furthermore, they do not resemble the effects of depolarization induced by perfusion with elevated K solutions. The cAMP-induced changes in growth cone morphology that we report here suggest a possible role for protein phosphorylation in promoting growth cone differentiation and structural changes accompanying secretion.

Movement of mitochondria in the ovarian trophic cord of Dysdercus intermedius (Heteroptera) resembles nerve axonal transport.

The motile behaviour of mitochondria in the ovarian trophic cord of the red cotton bug, Dysdercus intermedius, was observed optically using video-enhanced differential interference contrast (AVEC-DIC) microscopy. The motion of 258 video-recorded mitochondria was analysed of which 10%-30% were found to move during the observation periods. Of the moving mitochondria 76% travelled towards the oocyte with an average velocity of 3.37 μm/ min, and 24% towards the tropharium with 2.84 μm/min. The movement was found to be basically of the saltatory type I as known from nerve axons characterized by the absence of directional reversal. In some cases short periods of interrupted motion of type II, i.e. with local oscillations, were observed. Individual mitochondria often showed velocity variations during the excursions. The hemipteran trophic cords are known to contain numerous parallel microtubules. As the observed type of mitochondrial motility resembles axonal transport, a modified transport hypothesis is presented for the microtubule-based motility of organelles in the nurse strands of telotrophic insect ovarioles.

Glial-guided granule neuron migration in vitro: a high-resolution time- lapse video microscopic study

To study neuronal migration, migrating granule neurons in microcultures prepared from early postnatal cerebellum have been analyzed with time-lapse, video-enhanced differential interference contrast microscopy. The morphology of migrating neurons resembles the elongated forms of migrating neurons described both in vivo and in vitro (Rakic, 1971; Hatten et al., 1984). The neuron closely apposes its soma along the glial fiber and extends a thickened leading process in the direction of migration. This leading tip is highly motile, with several filopodial extensions. Intracellular vesicular structures extend from the nucleus into the leading process of migrating neurons in vitro. Quantitation of the motions of migrating neurons revealed a saltatory pattern of advance along the glial fiber. Periods of cell soma movement at the rate of 56 +/- 26 micron/hr along the glial fiber are punctuated by periods during which the cell soma slows to a complete stop. The overall rate of migration is 33 +/- 20 micron/hr. The growing tip of the leading process rapidly extends and retracts, resulting in a net advance along the glial fiber. However, the periods of the extension and retraction of the leading process growing tip are not synchronized with the motions of the cell soma.