This proposal addresses the uncharacterized role of the mInsc-LGN-Gai protein complex in stereocilia
elongation, and defines LGN-Gai as a long sought-after cargo of Myo15 required for differential stereocilia
growth across rows during hair cell development. In the absence of LGN or Gai, postnatal stereocilia are
stunted and form an immature-looking bundle that only retains a shallow staircase pattern. Interestingly, very
similar defects have been reported in the absence of Myo15, Whirlin and Eps8, three proteins enriched at the
tips of all stereocilia in amounts proportional to their height. We show here that LGN and Gai share the same
tip localization, but are restricted to the first stereocilia row. Strikingly, LGN and Gai fail to traffic to tips in
absence of Myo15. In contrast, Myo15-Whrn-Eps8 are found in similar low amounts across all stunted rows in
LGN or Gai mutants, suggesting that LGN-Gai instruct differential stereocilia identity across rows by specifying
the first row. By intercrossing mutants and using genetics, protein immunolocalization and protein binding
biochemistry, we propose to solve the function of an extended Myo15 complex now including LGN-Gai where
all proteins apparently have independent, complementary functions to shape the bundle.
We previously showed that mInsc-LGN-Gai first occupy and generate the 'bare zone', the flat region of the hair
cell apex where early microvilli exclusion outlines the V-shaped or semi-circular edge of the forming bundle.
Based on evidence for dynamic and balanced LGN-Gai protein amounts between subcellular compartments,
we propose that restriction of LGN-Gai to the first row could be instructed by their prior enrichment at the
adjacent bare zone. LGN-Gai would promote higher amounts of Myo15-Whrn-Eps8 at the first row, its growth
into the tallest row in turn influencing the height of shorter rows via oblique tip-links, as proposed previously. To
test the novel idea that the staircase pattern is established by recycling planar polarity information, we propose
stage-specific loss- and gain-of-function approaches, including exploratory experiments using cochlear
explants aimed at elucidating and manipulating LGN-Gai function at the subcellular level.
The staircase-like architecture of the hair bundle is essential for hearing and considered instrumental for
direction-sensitivity to sound stimuli, but remains largely unexplained at the molecular level. By uncovering new
members of the Myo15 complex and clarifying how asymmetry of growth is created across rows, this work
notably improves our understanding of hereditary hearing loss in DFNB3 (MYO15), DFNB31/USH2D (WHRN),
DFNB102 (EPS8) and DFNB82/Chudley-McCullough syndrome (LGN/GPSM2).