Beth Burnside

The mechanical basis of morphogenesis: I. Epithelial folding and invagination☆

We present a mechanical model for the morphogenetic folding of embryonic epithelia based on hypothesized mechanical properties of the cellular cytoskeleton. In our model we consider a simple cuboidal epithelium whose cells are joined at their apices by circumferential junctions; to these junctions are attached circumferential arrays of microfilament bundles assembled into a “purse string” around the cell apex. We assume that this purse string has the following property: if its circumference is increased beyond a certain threshold, an active contraction is initiated which “draws the purse-string” and reduces the apical circumference of the cell to a new, shorter, resting length. The remainder of the cell is modeled as a visoelastic body of constant volume. Clearly contraction in one cell could stretch the apical circumferences of neighboring cells and, if the threshold is exceeded, cause them “to fire” and contract. The objective of this paper is to demonstrate that our model, based on the local behavior of individual cells, generates a propagating contraction wave which is sufficient to explain the globally coherent morphogenetic infolding of a wide variety of embryonic epithelia. Representative computer simulations, based on the model, are presented for the initial gastrulation movements of echinoderms, neural tube formation in urodele amphibians, and ventral furrow formation in Drosophila.

Dopaminergic Regulation of Cone Retinomotor Movement in Isolated Teleost Retinas: I. Induction of Cone Contraction Is Mediated by D2 Receptors

Abstract: In the retinas of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) undergo characteristic movements in response to changes in light intensity and to signals from an endogenous circadian clock. To identify agents responsible for mediating light and/or circadian regulation of these retinomotor movements, we investigated the effects of hormones and neurotransmitters on cone, rod, and RPE movements in the green sunfish, Lepomis cyanellus. We report here that 3,4‐dihydroxyphenylethylamine (dopamine) mimics the effect of light by inducing light‐adaptive retinomotor movements in all three cell types. In isolated dark‐cultured retinas, dopamine induced light‐adaptive cone contraction with a halfmaximal effect at 10−8M. This effect of dopamine was inhibited by antagonists with a potency order characteristic of D2 receptor mediation. The dopamine uptake blocker benztropine also induced light‐adaptive cone contraction in isolated dark‐cultured retinas, suggesting that there is continuous dopamine release in the dark but that concomitant uptake normally prevents activation of cone contraction. That dopamine plays a role in light regulation of cone movement is further suggested by the observation that light‐induced cone contraction was partially inhibited by sulpiride, a selective D2 dopamine antagonist, or by Co2+, a blocker of synaptic transmission. Sulpiride also promoted dark‐adaptive cone elongation in isolated light adapted retinas, suggesting that continuous dopamine action is required in the light to maintain the light‐adapted cone position. Dopamine can act directly on D2 receptors located on rod and cone inner/outer segments: dopamine induced light‐adaptive retinomotor movements in isolated distal fragments of dark‐adapted photoreceptors cultured in the dark. Together our results indicate that dopamine induces light‐adaptive retinomotor movements in cones, rods, and RPE cells by activating D2 receptors. We suggest that, in vivo, dopamine plays a role in both light and circadian regulation of retinomotor movements.

A New Compartment at Stereocilia Tips Defined by Spatial and Temporal Patterns of Myosin IIIa Expression

Class III myosins are motor proteins that contain an N-terminal kinase domain and a C-terminal actin-binding domain. We show that myosin IIIa, which has been implicated in nonsyndromic progressive hearing loss, is localized at stereocilia tips. Myosin IIIa progressively accumulates during stereocilia maturation in a thimble-like pattern around the stereocilia tip, distinct from the cap-like localization of myosin XVa and the shaft localization of myosin Ic. Overexpression of deletion mutants for functional domains of green fluorescent protein (GFP)–myosin IIIa shows that the motor domain, but not the actin-binding tail domain, is required for stereocilia tip localization. Deletion of the kinase domain produces stereocilia elongation and bulging of the stereocilia tips. The thimble-like localization and the influence myosin IIIa has on stereocilia shape reveal a previously unrecognized molecular compartment at the distal end of stereocilia, the site of actin polymerization as well as operation of the mechanoelectrical transduction apparatus.

Dopamine Inhibits Forskolin- and 3-Isobutyl-1-Methylxanthine-Induced Dark-Adaptive Retinomotor Movements in Isolated Teleost Retinas

We have been investigating the mechanisms of diurnal and circadian regulation of teleost retinomotor movements. In the retinas of lower vertebrates, photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) undergo movements at dawn and dusk. These movements continue to occur at subjective dawn and dusk in animals maintained in constant darkness. Cone myoids contract at dawn and elongate at dusk; RPE pigment disperses into the epithelial cells’ long apical processes at dawn and aggregates into the cell bodies at dusk. We report here that forskolin, an adenylate cyclase activator, and 3‐isobutyl‐1‐methylxanthine (IBMX), a phosphodiesterase inhibitor, each induces dark‐adaptive cone and RPE retinomotor movements in isolated light‐adapted green sunfish retinas cultured in constant light. Forskolin induces a 22‐fold elevation in retinal cyclic AMP content. Forskolin‐ and IBMX‐induced movements are inhibited approximately 65% and 95%, respectively, by 3,4‐dihydroxyphenylethylamine (dopamine). However, dopamine does not inhibit dark‐adaptive movements induced by dibutyryl cyclic AMP. Epinephrine is much less effective than dopamine in inhibiting forskolin‐induced movements, while phenylephrine and clonidine are totally ineffective. These results are consistent with our previous findings that treatments that increase intracellular cyclic AMP content promote darkadaptive retinomotor movement. They further suggest that dopamine inhibits adenylate cyclase activity in photoreceptors and RPE cells and thereby favors light‐adaptive retinomotor movements.

Dopamine Induces Light-Adaptive Retinomotor Movements in Bullfrog Cones via D2 Receptors and in Retinal Pigment Epithelium via D1 Receptors

In the eyes of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) exhibit characteristic retinomotor movements in response to changes in ambient illumination and to signals from an endogenous circadian clock. We previously reported that 3,4‐dihydroxyphenylethylamine (dopamine) mimicked the effect of light on these movements in photoreceptors and RPE cells of green sunfish, Lepomis cyanellus, by interacting with D2 dopaminergic receptors. Here, we report that dopamine also mimics the effect of light on cone and RPE retinomotor movements in bullfrogs, Rana catesbeiana, i.e., dopamine induces cone contraction and RPE pigment dispersion. Dopamine induced cone contraction in isolated dark‐adapted bullfrog retinas incubated in constant darkness in the presence of the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX). This effect of dopamine was inhibited by a D2 but not a D1 antagonist and mimicked by a D2 but not a D1 agonist. These results suggest that induction of cone contraction by dopamine is mediated by D2 dopaminergic receptors and that cone adenylate cyclase activity is inhibited. Thus, dopamine acts via the same type of receptor in both bullfrog and green sunfish retinas to induce cone contraction. In contrast, dopamine influences RPE retinomotor movement via different receptors in fish and bullfrog. Dopamine induced light‐adaptive pigment dispersion in isolated dark‐adapted bullfrog RPE‐eyecups incubated in constant darkness in normal Ringers solution. Because the retina was not present, these experiments demonstrate a direct effect of dopamine on bullfrog RPE. This effect of dopamine on bullfrog RPE was inhibited by a D1 but not a D2 antagonist and mimicked by a D1 but not a D2 agonist. Furthermore, agents that increase the concentration of intracellular cyclic AMP also induced pigment dispersion in dark‐adapted bullfrog RPE‐eyecups incubated in the dark. These results suggest that dopamine induces pigment dispersion in bullfrog RPE via D1 dopaminergic receptors. Thus, dopamine acts via different receptors on bullfrog (D1) versus green sunfish (D2) RPE to induce pigment dispersion. In addition, inhibitor studies indicate that pigment dispersion is actin dependent in teleost but not in bullfrog RPE. Dopamine‐induced pigment dispersion was inhibited by cytochalasin D in isolated RPE sheets of green sunfish but not in RPE‐eyecups of bullfrogs. Together, these observations indicate that dopamine mimics the effect of light on cone and RPE retinomotor movements in both fish and bullfrogs. However, in the RPE, different receptors mediate the effect of dopamine, and different cytoskeletal mechanisms are used to affect pigment transport. These findings suggest that the association of dopamine release with light onset is of earlier evolutionary origin than the appearance of retinomotor movements and that retinomotor movements may have evolved separately in teleosts and amphibians.

Stimulation of Distinct D2 Dopaminergic and α2-Adrenergic Receptors Induces Light-Adaptive Pigment Dispersion in Teleost Retinal Pigment Epithelium

Abstract: In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules aggregate and disperse in response to changes in light conditions. Pigment granules aggregate into the RPE cell body in the dark and disperse into the long apical projections in the light. Pigment granule movement retains its light sensitivity in vitro only if RPE is explanted together with neural retina. In the absence of retina, RPE pigment granules no longer move in response to light onset or offset. Using a preparation of mechanically isolated fragments of RPE from green sunfish, Lepomis cyanellus, we investigated the effects of catecholamines on pigment migration. We report here that 3,4‐dihydroxyphenylethylamine (dopamine) and clonidine each mimic the effect of light in vivo by inducing pigment granule dispersion. Dopamine had a half‐maximal effect at approximately 2 nM; clonidine, at 1 μM. Dopamine‐induced dispersion was inhibited by the D2 dopaminergic antagonist sulpiride but not by Dl or α‐adrenergic antagonists. Furthermore, a D2 dopaminergic agonist (LY 171555) but not a Dl dopaminergic agonist (SKF 38393) mimicked the effect of dopamine. Clonidine‐induced dispersion was inhibited by the α2‐adrenergic antagonist yohimbine but not by sulpiride. These results suggest that teleost RPE cells possess distinct D2 dopaminergic and α2‐adrenergic receptors, and that stimulation of either receptor type is sufficient to induce pigment granule dispersion. In addition, forskolin, an activator of adenylate cyclase, induced pigment granule movement in the opposite direction, i.e., dark‐adaptive pigment aggregation. Together, our results suggest that stimulation of D2 dopaminergic or α2‐adrenergic receptors on teleost RPE induces light‐adaptive pigment granule dispersion at least in part by inhibiting adenylate cyclase activity in these cells. We suggest that modulation of RPE cAMP levels by catecholamines supplied from either the retina or the choroid could contribute to the regulation of RPE responses to both light and circadian signals.

Kinetic Mechanism of Human Myosin IIIA

Myosin IIIA is specifically expressed in photoreceptors and cochlea and is important for the phototransduction and hearing processes. In addition, myosin IIIA contains a unique N-terminal kinase domain and C-terminal tail actin-binding motif. We examined the kinetic properties of baculovirus expressed human myosin IIIA containing the kinase, motor, and two IQ domains. The maximum actin-activated ATPase rate is relatively slow (kcat = 0.77 ± 0.08 s–1), and high actin concentrations are required to fully activate the ATPase rate (KATPase = 34 ± 11 μm). However, actin co-sedimentation assays suggest that myosin III has a relatively high steady-state affinity for actin in the presence of ATP (Kactin ∼ 7 μm). The rate of ATP binding to the motor domain is quite slow both in the presence and absence of actin (K1k+2 = 0.020 and 0.001 μm–1·s–1, respectively). The rate of actin-activated phosphate release is more than 100-fold faster (85 s–1) than the kcat, whereas ADP release in the presence of actin follows a two-step mechanism (7.0 and 0.6 s–1). Thus, our data suggest a transition between two actomyosin-ADP states is the rate-limiting step in the actomyosin III ATPase cycle. Our data also suggest the myosin III motor spends a large fraction of its cycle in an actomyosin ADP state that has an intermediate affinity for actin (Kd ∼ 5 μm). The long lived actomyosin-ADP state may be important for the ability of myosin III to function as a cellular transporter and actin cross-linker in the actin bundles of sensory cells.

Adenosine Stimulates Cone Photoreceptor Myoid Elongation via an Adenosine A2‐Like Receptor

Abstract: In several parts of the nervous system, adenosine has been shown to function as an extracellular neuromodulator binding to surface receptors on target cells. This study examines the possible role of adenosine in mediating light and circadian regulation of retinomotor movements in teleost cone photoreceptors. Teleost cones elongate in the dark and contract in the light. In continuous darkness, the cones continue to elongate and contract at subjective dusk and dawn in response to circadian signals. We report here that exogenous adenosine triggers elongation (the dark/night movement) in isolated cone inner segment‐cone outer segment preparations (CIS‐COS) in vitro. Agonist/antagonist potency profiles indicate that adenosines effect on cone movement is mediated by an A2‐like adenosine receptor, which like other A2 receptors enhances adenylate cyclase activity. Although closest to that expected for A2 receptors, the antagonist potency profile for CIS‐COS does not correspond exactly to any known A2 receptor subtype, suggesting that the cone receptor may be a novel A2 subtype. Our findings are consistent with previous reports that retinal adenosine levels are higher in the dark, and further suggest that adenosine could act as a neuromodulatory “dark signal” influencing photoreceptor metabolism and function in the fish retina.

A role for endogenous dopamine in circadian regulation of retinal cone movement

Cone movements in the retina of the Midas cichlid (Cichlasoma citrinellum) take place in response both to light and endogenous circadian signals. In the normal light/dark cycle (LD) cone myoids are long at night (50-55 microns), begin to contract before expected dawn, and with light onset contract to their fully contracted positions (5 microns) which are retained throughout the day. In continuous darkness (DD) cone myoids are fully elongate at night, but undergo pre-dawn contractions to partially contracted positions which they retain throughout expected day (20-25 microns). To investigate the mechanisms by which circadian signals modulate cone myoid movements in teleost retinas, we have tested the effects on circadian cone movements of optic nerve section, intraocular injection of dopamine agonists or antagonists, and intraocular injection of melatonin. We report here that both light-induced and circadian-driven cone myoid movements can occur in the absence of efferent input from higher centres: both are retained with full amplitude after optic nerve section in vivo. Intraocular injection studies suggest that circadian regulation of cone myoid movement is mediated locally within the eye by dopamine acting via a dopaminergic D2-receptor. Cone myoid contraction can be induced at midnight in LD or DD animals by intraocular injection of dopamine or the D2-receptor agonist LY171555. The partially contracted cones of DD animals at expected mid-day can be induced to fully contract by intraocular injection of dopamine or the D2-receptor agonist, or to elongate by intraocular injection of the dopamine D2-antagonist sulpiride. Furthermore, the pre-dawn cone myoid contraction observed in both LD and DD animals in response to circadian signals can be completely blocked in DD animals by intraocular injection of the D2-antagonist sulpiride shortly before the time of expected light onset. In contrast, circadian cone myoid movements were unaffected by intraocular injection of the D1-receptor agonist SCH23390, or the D1-receptor antagonist SKF38393. In addition, we report that intraocularly injected melatonin had no effect on cone position when injected in the light at mid-day, in darkness at midnight or in darkness just before expected light onset at dawn. However, both melatonin and iodomelatonin induced cone myoid contraction (the light-adaptive movement) when injected in darkness at expected mid-day in DD animals. This paradoxical result is not consistent with observations from other species in which melatonin induces dark-adaptive photoreceptor responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Microtubule nucleation and organization in teleost photoreceptors : microtubule recovery after elimination by cold

SummaryRetinal photoreceptors have two separate populations of microtubules: axonemal microtubules of the modified cilium of the outer segment and cytoplasmic microtubules of the cell body. The axonemal microtubules originate from a basal body located at the distal tip of the photoreceptor inner segment and extend in a 9+0 configuration into the outer segment of rods and accessory outer segment of cones. The cytoplasmic microtubules of the cell body are axially aligned from the distal tip of the inner segment to proximal synapse, and are oriented with uniform polarity, their minus ends distal toward the outer segment and plus ends proximal toward the synapse (Troutt & Burnside, 1988). To investigate how this regular cytoplasmic microtubule array is generated, we have attempted to identify microtubule nucleation sites in the cones of the tropical teleost fish, Tilapia (Sarotherodon mossambicus) by examining the regrowth of cytoplasmic microtubules after cold disruption in whole retinas or in isolated cone fragments consisting of inner and outer segments (CIS-COS). Incremental stages of microtubule reassembly were examined both by electron microscopy of thin sections and by immunofluorescent localization of microtubules with an antitubulin antibody. Cold treatment completely abolished all cytoplasmic microtubules but did not disrupt axonemal microtubules. Within 2 min after rewarming, cytoplasmic microtubules reappeared in the most distal portion of the inner segment in a small aster-like array associated with the basal body, and subsequently appeared in more proximal parts of the cone. These observations suggest that a favoured microtubule nucleation site is associated with the basal body region of the cone outer segment, and thus that the basal body region could function as a microtubule organizing centre for the photoreceptor. These results are consistent with the findings of our previous investigation of cone microtubule polarity, which showed that the minus ends of the cytoplasmic microtubules of the cone are associated with the basal body region.