lhfpl5a (LHFPL tetraspan subfamily member 5a) is one of two zebrafish co-orthologs of vertebrate LHFPL5 (TMHS), retained from the teleost-specific genome duplication; its paralog is lhfpl5b. It encodes a small four-pass membrane protein of the tetraspan junctional protein superfamily that acts as an accessory subunit of the hair-cell mechanoelectrical transduction (MET) complex. GFP-Lhfpl5a sits at the tips of stereocilia and is enriched at the tallest stereocilia next to the kinocilium; its bundle localization depends on Pcdh15a, Cdh23 and Myo7aa, and Lhfpl5a in turn is needed to bring the tip-link protein Pcdh15a to the hair bundle. It is not needed for bundle targeting of Tmc1 or Tmc2b. lhfpl5a is expressed only in hair cells of the inner ear (maculae and cristae), from 1 dpf into adulthood, and not in the lateral line. The astronaut (tm290d) nonsense mutant has no transduction in any inner-ear sensory patch and is deaf and balance-defective, with splayed hair bundles, while lateral-line hair cells, which use lhfpl5b, work normally.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Plasma membrane location propagated across the LHFPL family. Reason: Lhfpl5a is a four-pass membrane protein and GFP-Lhfpl5a is found at stereocilia tips, in the plasma membrane of the hair bundle. Correct but general; the stereocilium tip is proposed as a NEW, more informative location. Supporting Evidence: PMID:32009898 GFP-Lhfpl5a is present at the tips of stereocilia, similar to the localization observed for mouse LHFPL5 (Xiong et al., 2012; Mahendrasingam et al., 2017). |
| GO:0007605 sensory perception of sound | IBA GO_REF:0000033 | ACCEPT | Summary: Sensory perception of sound, from the PANTHER LHFPL5 node (mouse and fly donors). Reason: lhfpl5a is the copy expressed in the inner ear, and lhfpl5a mutants are profoundly deaf; the GFP-Lhfpl5a transgene rescues the acoustic startle reflex. Lhfpl5a contributes through transduction, captured more specifically by GO:0050974. Supporting Evidence: PMID:32009898 Results confirm that lhfpl5atm290d mutants are profoundly deaf, displaying little to no response to acoustic stimuli (p < 0.001 compared to all other genotypes). PMID:32009898 From these results we conclude that the hair bundle-localized GFP-Lhfpl5a protein is functional and can rescue the behavioral and MET channel defects in lhfpl5atm290d mutants. |
| GO:0016020 membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Membrane, from UniProt subcellular location (multi-pass membrane protein). Reason: Correct; less specific than the plasma membrane and stereocilium tip locations. |
| GO:0050974 detection of mechanical stimulus involved in sensory perception | IBA GO_REF:0000033 | ACCEPT | Summary: Detection of mechanical stimulus involved in sensory perception, from the PANTHER LHFPL5 node. Reason: Core process. lhfpl5a mutants lose transduction in all sensory patches of the ear, and the tagged protein rescues it. The WITH list includes lhfpl5a itself; that is expected, because its own experimental data are among the descendant evidence for the node. Supporting Evidence: PMID:32009898 These three tests confirmed that all sensory patches in the otic capsule are inactive in lhfpl5atm290d mutants. PMID:32009898 From these results we conclude that the hair bundle-localized GFP-Lhfpl5a protein is functional and can rescue the behavioral and MET channel defects in lhfpl5atm290d mutants. |
| GO:0050974 detection of mechanical stimulus involved in sensory perception | IMP PMID:9491988 Genetic analysis of vertebrate sensory hair cell mechanosens... | ACCEPT | Summary: From the original characterization of the astronaut (lhfpl5a) circler mutant, which is defective in balance and hearing. Reason: The 1998 abstract placed astronaut downstream of transduction because lateral-line microphonics were normal. Later work explains this: lateral-line hair cells use lhfpl5b, whereas all inner-ear sensory patches of lhfpl5a mutants lack transduction. The annotated process is therefore correct for lhfpl5a, and the curator's call is upheld by the later data. Supporting Evidence: PMID:9491988 Mutant astronaut and cosmonaut hair cells have relatively normal microphonics and thus appear to affect events downstream of mechanotransduction. PMID:32009898 lhfpl5atm290d (astronaut/asn) mutants were initially characterized by balance defects, an absence of the acoustic startle reflex, and a lack of brainstem Ca2+ signals after acoustic startle. However, neuromast microphonic potentials were normal (Nicolson et al., 1998). PMID:32009898 These three tests confirmed that all sensory patches in the otic capsule are inactive in lhfpl5atm290d mutants. |
| GO:0060122 inner ear receptor cell stereocilium organization | IMP PMID:32009898 The lhfpl5 Ohnologs lhfpl5a and lhfpl5b Are Required for Mec... | KEEP AS NON CORE | Summary: Inner ear receptor cell stereocilium organization: lhfpl5a mutant inner-ear hair bundles are splayed. Reason: The phenotype is real. The most direct reading is that it follows from the failure to bring Pcdh15a, and so tip links, to the bundle; Lhfpl5a is not known to organize stereocilia by itself. Kept as a correct but non-core consequence of its role in the MET/tip-link complex. Supporting Evidence: PMID:32009898 lhfpl5atm290d mutants exhibit splayed hair bundles, with low levels of Pcdh15a staining restricted to the tip of each stereocilium PMID:28219986 In contrast, EGFP-tagged Pcdh15a remained in the hair cell body of lhfpl5a mutants at all developmental stages, implying that Lhfpl5a is required for transport to the hair bundle. |
| GO:0061512 protein localization to cilium | IMP PMID:28219986 Functional Analysis of the Transmembrane and Cytoplasmic Dom... | MODIFY | Summary: Protein localization to cilium: Pcdh15a fails to reach the hair bundle in lhfpl5a mutants. Reason: The observation is sound: tagged and endogenous Pcdh15a stay in the cell body of lhfpl5a mutant hair cells, and the authors infer that Lhfpl5a is needed for transport to the hair bundle. But the destination is the stereocilia, which are actin-based microvillar projections and not cilia; GO:0032420 stereocilium is not a descendant of cilium. GO has no protein-localization-to-stereocilium term, so the nearest correct term is protein localization to plasma membrane (Pcdh15a is a transmembrane tip-link protein that must reach the stereocilia membrane). Proposed replacements: protein localization to plasma membrane Supporting Evidence: PMID:28219986 We next examined the localization pattern of stably expressed Pcdh15a-CD3-GFP in lhfpl5a tm290d mutants and found that Pcdh15a-CD3-EGFP was largely absent in both mature and immature hair bundles, but detectable in hair cell somas PMID:28219986 In contrast, EGFP-tagged Pcdh15a remained in the hair cell body of lhfpl5a mutants at all developmental stages, implying that Lhfpl5a is required for transport to the hair bundle. |
| GO:0032426 stereocilium tip | IDA PMID:32009898 The lhfpl5 Ohnologs lhfpl5a and lhfpl5b Are Required for Mec... | NEW | Summary: A functional GFP-Lhfpl5a transgene localizes to the tips of stereocilia in inner-ear hair cells. Reason: Direct localization of a tagged protein shown to rescue the mutant, matching mouse LHFPL5. This is the site of action and is more informative than the plasma membrane rows. Supporting Evidence: PMID:32009898 GFP-Lhfpl5a is present at the tips of stereocilia, similar to the localization observed for mouse LHFPL5 (Xiong et al., 2012; Mahendrasingam et al., 2017). PMID:32009898 From these results we conclude that the hair bundle-localized GFP-Lhfpl5a protein is functional and can rescue the behavioral and MET channel defects in lhfpl5atm290d mutants. |
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Download this section (compressed HTML)Q: Does Lhfpl5b rescue lhfpl5a mutant inner-ear hair cells when expressed there? The reverse rescue (Lhfpl5a in lhfpl5b mutant neuromasts) works; the reciprocal would complete the evidence that the two proteins are interchangeable.
Q: Is lhfpl5b ever expressed in adult inner-ear hair cells, or is the ear/lateral-line split maintained for life?
Q: What protein links Lhfpl5a to Cdh23 and Myo7aa for its localization at kinocilial links, given that no direct LHFPL5-CDH23 interaction has been detected?
Experiment: Express GFP-Lhfpl5b from a pan-hair-cell (myo6b) or lhfpl5a promoter in lhfpl5a tm290d mutants and score acoustic startle, inner-ear microphonics and FM dye uptake.
Hypothesis: The lhfpl5a/lhfpl5b split is purely regulatory.
Type: cross-rescue
Experiment: Test gar lhfpl5 upstream sequences in zebrafish reporter assays for activity in both inner-ear and lateral-line hair cells; compare with the lhfpl5b 2 kb promoter that already drives lateral-line-specific expression.
Hypothesis: The cis-regulatory elements that restrict lhfpl5a to the ear and lhfpl5b to the lateral line are ancestral elements partitioned between the copies.
Type: enhancer reporter assay
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