3D printed chamber for live cell imaging on an upright epifluorescence microscope.

Live cell imaging is a standard technique in experimental biology that enables the observation of isolated cells and tissue slices in real time; and the testing of cellular responses to changes in buffer composition. However, most live cell imaging devices require the use of dedicated microscopes and/or specialized stage adaptors, and come at a reasonably high cost. We employed 3D printing technology to create a low-cost imaging chamber with side ports to exchange fluids, to be used on upright microscopes. The chamber increased the functionality of a standard upright epifluorescent microscope to allow dynamic, real-time calcium imaging of cultured hypothalamic astrocytes from mice, and to test the effects of ATP stimulation upon calcium signaling. It was also used on slices obtained from mouse brain using a brain matrix slicer. The advantages of this chamber include a very simple design that can be used with upright epifluorescence microscopes, does not require any special stage adaptor, and includes ports to permit fluid exchange during imaging. This chamber is ideal for educational settings with undergraduate laboratories that do not have access to dedicated inverted fluorescent microscopes for tissue culture experiments.

Rho-family G-proteins are required for the recovery of traumatized hair bundle mechanoreceptors in the sea anemone, Nematostella vectensis.

Immersing anemones in calcium-free seawater disorganizes hair bundle mechanoreceptors on tentacles of sea anemones while causing a loss of vibration sensitivity. Remarkably, anemone hair bundles recover after being returned to calcium-containing seawater. Reorganization of actin in stereocilia likely follows during the recovery of normal morphology of hair bundles after such immersion. Previous studies have reported that Rho G-proteins are located in the stereocilia of hair bundles in sea anemones where they participate in polymerizing actin in stereocilia upon activation of specific chemoreceptors. We here find that immersing anemones in calcium-free seawater significantly reduces the abundance of hair bundles. A partial recovery of abundance of hair bundles occurs within 3 h post-immersion, but a full recovery of abundance does not occur even 6 h after specimens are returned to calcium-containing seawater. Anemones recovering from immersion in calcium-free seawater feature hair bundles that are significantly wider at their tips than in controls. The hair bundles subsequently narrow at their tips, becoming comparable to those of untreated controls within 6 h. Stereocilia of hair bundles are significantly longer in experimental animals than in controls at 2 h of recovery before shortening to lengths comparable to untreated controls at 6 h. In the presence of Rho inhibitors, the recovery in abundance of hair bundles through 6 h is delayed or inhibited. Likewise, in the presence of Rho inhibitors, stereocilia fail to significantly elongate within 2 h of recovery. These data suggest that Rho G-proteins participate in the normal recovery of abundance and recovery of normal morphology of experimentally damaged hair bundle mechanoreceptors.

Self/Non-Self Recognition Affects Cnida Discharge and Tentacle Contraction in the Sea Anemone Haliplanella luciae.

Certain species of sea anemone live in tightly packed communities, among clonemates and non-clonemates. Competition for space leads to intraspecific and interspecific aggressive interactions among anemones. The initial aggressive interactions appear to involve reciprocal discharge of cnidae triggered by contact with non-self feeding tentacles. We asked whether molecules contained in anemone-derived mucus constituted an important cue alone or in combination with cell surface molecules in stimulating aggressive or avoidance behaviors. In this study, we found that self and non-self stimuli differentially influenced two effector systems: cnida discharge and tentacle contraction. Interspecific mucus enhanced nematocyst discharge by 44% and spirocyst discharge by 90%, as compared to baseline discharge obtained in seawater alone. Conspecific stimuli accompanying touch inhibited specific tentacle contractions occurring on the far side of anemones relative to the site of contact. The greatest tentacle contractions occurred with exposure to interspecific mucus and tissue. Thus, several receptor systems are involved that integrate chemical and mechanical cues in order to initiate appropriate and graded effector responses during competition for space.

Evidence for two populations of hair bundles in the sea anemone, Nematostella vectensis.

Cytochalasin D (CD) was employed to disrupt F-actin within stereocilia of anemone hair bundles. CD treatment decreases the abundance of hair bundles (by 85%) while significantly impairing predation. The remaining hair bundles are 'CD-resistant.' Surprisingly, the morphology and F-actin content of resistant hair bundles are comparable to those of untreated controls. However, the resistant hair bundles fail to respond normally to the N-acetylated sugar, NANA, by elongating. Instead, they remain at resting length. Immediately after CD treatment, when only CD-resistant hair bundles are present, nematocyst discharge is normal into targets touched to tentacles in the absence of vibrations (i.e., baseline) but fails to increase normally in the presence of nearby vibrations at 56Hz, a key frequency. After CD treatment, the abundance of hair bundles recovers to control levels within three hours. At 2h after CD treatment, when CD-resistant and CD-sensitive hair bundles are both present, but a full-recovery is not yet complete, somewhat enhanced discharge of nematocysts occurs into targets touched to tentacles in the presence of nearby vibrations at 56Hz (at least as compared to the response of CD-treated animals to contact with test probes in the absence of vibrations). Additionally, at 2h after CD-treatment, prey capture recovers to normal. Thus, two populations of hair bundles may be present on tentacles of sea anemones: those that are CD-resistant and those that are CD-sensitive. The functions of these hair bundles may be distinct.

Repair of traumatized mammalian hair cells via sea anemone repair proteins.

Mammalian hair cells possess only a limited ability to repair damage after trauma. In contrast, sea anemones show a marked capability to repair damaged hair bundles by means of secreted repair proteins (RPs). Previously, it was found that recovery of traumatized hair cells in blind cavefish was enhanced by anemone-derived RPs; therefore, the ability of anemone RPs to assist recovery of damaged hair cells in mammals was tested here. After a 1 h incubation in RP-enriched culture media, uptake of FM1-43 by experimentally traumatized murine cochlear hair cells was restored to levels comparable to those exhibited by healthy controls. In addition, RP-treated explants had significantly more normally structured hair bundles than time-matched traumatized control explants. Collectively, these results indicate that anemone-derived RPs assist in restoring normal function and structure of experimentally traumatized hair cells of the mouse cochlea.

Proteomic identification of hair cell repair proteins in the model sea anemone Nematostella vectensis.

Sea anemones have an extraordinary capability to repair damaged hair bundles, even after severe trauma. A group of secreted proteins, named repair proteins (RPs), found in mucus covering sea anemones significantly assists the repair of damaged hair bundle mechanoreceptors both in the sea anemone Haliplanella luciae and the blind cavefish Astyanax hubbsi. The polypeptide constituents of RPs must be identified in order to gain insight into the molecular mechanisms by which repair of hair bundles is accomplished. In this study, several polypeptides of RPs were isolated from mucus using blue native PAGE and then sequenced using LC-MS/MS. Thirty-seven known polypeptides were identified, including Hsp70s, as well as many polypeptide subunits of the 20S proteasome. Other identified polypeptides included those involved in cellular stress responses, protein folding, and protein degradation. Specific inhibitors of Hsp70s and the 20S proteasome were employed in experiments to test their involvement in hair bundle repair. The results of those experiments suggested that repair requires biologically active Hsp70s and 20S proteasomes. A model is proposed that considers the function of extracellular Hsp70s and 20S proteasomes in the repair of damaged hair cells.

Cadherin-23 may be dynamic in hair bundles of the model sea anemone Nematostella vectensis.

Cadherin 23 (CDH23), a component of tip links in hair cells of vertebrate animals, is essential to mechanotransduction by hair cells in the inner ear. A homolog of CDH23 occurs in hair bundles of sea anemones. Anemone hair bundles are located on the tentacles where they detect the swimming movements of nearby prey. The anemone CDH23 is predicted to be a large polypeptide featuring a short exoplasmic C-terminal domain that is unique to sea anemones. Experimentally masking this domain with antibodies or mimicking this domain with free peptide rapidly disrupts mechanotransduction and morphology of anemone hair bundles. The loss of normal morphology is accompanied, or followed by a decrease in F-actin in stereocilia of the hair bundles. These effects were observed at very low concentrations of the reagents, 0.1-10 nM, and within minutes of exposure. The results presented herein suggest that: (1) the interaction between CDH23 and molecular partners on stereocilia of hair bundles is dynamic and; (2) the interaction is crucial for normal mechanotransduction and morphology of hair bundles.

Rho participates in chemoreceptor-induced changes in morphology to hair bundle mechanoreceptors of the sea anemone, Nematostella vectensis.

Adjustable hair bundle mechanoreceptors located on anemone tentacles detect movements of nearby, swimming prey. The hair bundles are formed by numerous actin-based stereocilia that converge onto a single, central kinocilium. Interestingly, morphological and functional changes to the hair bundles are induced by activating chemoreceptors that bind prey-derived N-acetylated sugars and proline, respectively. Morphological changes to the hair bundles involve alterations to the actin cytoskeleton of stereocilia. A pharmacological activation of Rho induces hair bundles to elongate to lengths comparable to those normally induced by exposure to N-acetylneuraminic acid (NANA) and prevents shortening of hair bundles normally induced by proline. Rho inhibition prevents NANA-induced elongation, but does not prevent proline-induced shortening of hair bundles. Western blots feature a band similar in mass to that predicted for a Rho homolog in the genome of Nematostella. Immunocytochemistry localizes Rho in stereocilia of the hair bundle. Anemone hair bundles arise from multicellular complexes. Data from experiments using heptanol, a gap junction uncoupler, indicate that cell-cell communication is required in order for activated chemoreceptors to induce morphological changes to the hair bundles.

Force-dependent discharge of nematocysts in the sea anemone Haliplanella luciae (Verrill).

Sea anemones discharge cnidae ('stinging capsules' including nematocysts) to capture prey and to defend themselves. In the present study, we tested the relationship between the force of test probes striking feeding tentacles and discharge of microbasic p-mastigophore nematocysts into the test probes. In seawater alone, the response curve is bimodal with maximal discharge observed at 0.33 and 1.10 millinewtons (mN) and with minimal discharge at 1.50 mN. Upon activating chemoreceptors for N-acetylated sugars, maximal discharge is observed across a broad range of smaller forces from 0.16 to 0.9 mN before decreasing to a minimum at 1.50 mN. Likewise, in the presence of nearby vibrations at key frequencies, maximal discharge is observed over a broad range of smaller forces before decreasing to a minimum at 1.50 mN. It appears that sensory input indicating proximity of potential prey expands the range of small forces of impact that stimulate maximal discharge (i.e. to less than 1.10 mN) but not at larger forces of impact (i.e. at approximately 1.50 mN). Thus, contact by small prey would stimulate maximal discharge, and all the more so if such contact is accompanied by specific odorants or by vibrations at specific frequencies. Nevertheless, anemones would not maximally discharge nematocysts into large animals that blunder into contact with their tentacles.

Rheotaxis in larval zebrafish is mediated by lateral line mechanosensory hair cells.

The lateral line sensory system, found in fish and amphibians, is used in prey detection, predator avoidance and schooling behavior. This system includes cell clusters, called superficial neuromasts, located on the surface of head and trunk of developing larvae. Mechanosensory hair cells in the center of each neuromast respond to disturbances in the water and convey information to the brain via the lateral line ganglia. The convenient location of mechanosensory hair cells on the body surface has made the lateral line a valuable system in which to study hair cell damage and regeneration. One way to measure hair cell survival and recovery is to assay behaviors that depend on their function. We built a system in which orientation against constant water flow, positive rheotaxis, can be quantitatively assessed. We found that zebrafish larvae perform positive rheotaxis and that, similar to adult fish, larvae use both visual and lateral line input to perform this behavior. Disruption or damage of hair cells in the absence of vision leads to a marked decrease in rheotaxis that recovers upon hair cell repair or regeneration.

Evidence for involvement of TRPA1 in the detection of vibrations by hair bundle mechanoreceptors in sea anemones.

A homolog of TRPA1 was identified in the genome of the anemone, Nematostella vectensis (nv-TRPA1a), and predicted to possess six ankyrin repeat domains at the N-terminus and an ion channel domain near the C-terminus. Transmembrane segments of the ion channel domain are well conserved among several known TRPA1 polypeptides. Inhibitors of TRPA1 including ruthenium red decrease vibration-dependent discharge of nematocysts in N. vectensis and Haliplanella luciae. Activators of TRPA1 including URB-597 and polygodial increase nematocyst discharge in the absence of vibrations. Co-immunoprecipitation yields a band on SDS-PAGE gels at the predicted mass of the nv-TRPA1a polypeptide among other bands. Co-immunoprecipitation performed in the presence of antigenic peptide decreases the yield of this and several other polypeptides. In untreated controls, anti-nv-TRPA1a primarily labels the base of the hair bundle with some labeling also distributed along the length of stereocilia. Tissue immunolabeled in the presence of the antigenic peptide exhibits reduced labeling. Activating chemoreceptors for N-acetylated sugars induce immunolabel to distribute distally in stereocilia. In anemones, activating chemoreceptors for N-acetylated sugars induce hair bundles to elongate among several other structural and functional changes. Taken together, these results are consistent with the possibility that nv-TRPA1a participates in signal transduction of anemone hair bundles.

Cadherin 23-like polypeptide in hair bundle mechanoreceptors of sea anemones.

We investigated hair bundle mechanoreceptors in sea anemones for a homolog of cadherin 23. A candidate sequence was identified from the database for Nematostella vectensis that has a shared lineage with vertebrate cadherin 23s. This cadherin 23-like protein comprises 6,074 residues. It is an integral protein that features three transmembrane alpha-helices and a large extracellular loop with 44 contiguous, cadherin (CAD) domains. In the second half of the polypeptide, the CAD domains occur in a quadruple repeat pattern. Members of the same repeat group (i.e., CAD 18, 22, 26, and so on) share nearly identical amino acid sequences. An affinity-purified antibody was generated to a peptide from the C-terminus of the cadherin 23-like polypeptide. The peptide is expected to lie on the exoplasmic side of the plasma membrane. In LM, the immunolabel produced punctate fluorescence in hair bundles. In TEM, immunogold particles were observed medially and distally on stereocilia of hair bundles. Dilute solutions of the antibody disrupted vibration sensitivity in anemones. We conclude that the cadherin 23-like polypeptide likely contributes to the mechanotransduction apparatus of hair bundle mechanoreceptors of anemones.

Repair of hair cells following mild trauma may involve extracellular chaperones.

Sea anemones were subjected to mild trauma consisting of a 2 min immersion in calcium-depleted seawater. The trauma caused a loss of vibration sensitivity that spontaneously recovered within 50 min of returning the anemones to calcium containing seawater. Apparently, recovery is conferred by proteins contained in fraction gamma, a chromatographic fraction of homogenized mucus collected at the base of anemones allowed to recover from similar trauma. On silver stained SDS-PAGE gels, fraction gamma consists of a single band having an estimated mass of 55 kDa. Fraction gamma is alone sufficient to repair hair bundle mechanoreceptors in anemones. Its biological activity is enhanced in the presence of exogenously supplied ATP, but not GTP nor ADP-ribose. Biotinylated fraction gamma binds to hair bundles. The hypothesis that fraction gamma consists of Hsp60 proteins was tested. Commercial antibodies to Hsp60 label a band at 55 kDa in western blots. Hsp60 antibodies label hair bundles in traumatized anemones but not in untreated controls. Dilute Hsp60 antiserum (but not nonimmune serum) delays the spontaneous recovery of vibration sensitivity in anemones subjected to mild trauma. Thus, fraction gamma likely consists of Hsp60, or a Hsp60-like protein, that functions on the extracellular face of the plasma membrane to restore function to traumatized hair bundles.

The involvement of arl-5b in the repair of hair cells in sea anemones.

The subcellular processes involved in repair of hair cells are not well understood. Sea anemones repair hair bundle mechanoreceptors on their tentacles after severe trauma caused by 1-h exposure to calcium-depleted seawater. Repair is dependent on the synthesis and secretion of large protein complexes named "repair proteins." A cDNA library on traumatized anemone tissue was probed using polyclonal antibodies raised to a specific chromatographic fraction of the repair protein mixture. An ADP-ribosylation factor-like protein, Arl-5b, was identified. The amino acid sequence of the Arl-5b protein in sea anemones is similar to that among several model vertebrates and humans. A polyclonal antibody raised to a peptide of the anemone Arl-5b labels some but not all hair bundles in healthy control animals. The abundance of labeled hair bundles significantly increases above healthy controls after trauma and continuing through the first hour of recovery. Dilute anti-Arl-5b blocks the spontaneous repair of hair bundle mechanoreceptors, suggesting that Arl-5b acts on the extracellular face of the plasma membrane. Immunoelectron microscopy indicates that Arl-5b is located along the length of stereocilia including sites in the vicinity of tip links. We propose that Arl-5b is involved in installing replacement linkages into damaged hair bundle mechanoreceptors.

Rapid recovery of sensory function in blind cave fish treated with anemone repair proteins.

Blind cave fish employ superficial neuromasts to detect currents [Baker, C.F. and J.C. Montgomery, J. Comp. Physiol. A 184 (1999) 519-527]. Briefly exposing fish to calcium-free water significantly reduces the ability of the fish to perform rheotaxis (i.e., to orient properly in currents). Spontaneous recovery to control levels of rheotaxis requires 9 days. However, if the fish are treated with fraction beta immediately after exposure to calcium-free water, recovery to control levels of rheotaxis occurs within 1.3 h, the first time point tested. Fraction beta is a chromatographic fraction of 'repair proteins' isolated from sea anemones. The benefits of fraction beta on restoring rheotaxis exhibit dose dependency with the minimum effective dose estimated at 1 ng/ml. Exogenously supplied ATP augments the efficacy of fraction beta. Such augmentation is abolished by PPADS, an inhibitor of purinoceptors. Immunocytochemistry confirms the presence of purinoceptors in superficial neuromasts. The present results suggest that 'repair proteins' obtained from anemones significantly augment intrinsic repair mechanisms in fish. Furthermore, the data obtained in the fish system strongly parallel our previously published findings on sea anemones, raising the possibility that mechanisms of hair bundle repair may be evolutionarily conserved.