1. # 0064 Actin-Binding Protein 12 as a Component
of the Hair Cell’s Cuticular Plate and Stereocilia
1 University School, Hunting Valley Campus, 2Department of Genetics, 3Department of Otolaryngology – Head and Neck
Surgery, 4Department of Neurosciences, 5Department of Biology, Case Western Reserve University Medical School, Cleveland,
Ohio 44106
Andrew E. Megerian1, Lana M. Pollock2,3, and Brian M. McDermott Jr.2,34,5
INTRODUCTION:
The hair cells of the inner ear are auditory sensory receptors responsible for
converting sound waves into electrical impulses. The hair cell has distinguishing features,
which include the hair bundle and the cuticular plate. This specialized cell of the inner ear is
present in all vertebrates, from humans to fish. Because the structure and role of the hair cell
is conserved among species, studies in the fish are relevant to human hearing and deafness.
Especially pertinent is the role of actin-shaping proteins to the stereocilia and underlying
cuticular plate, considering these structures are comprised of primarily filamentous actin (F-
Actin). The conservation of the cuticular plate and stereocilia among species allows us to
better comprehend and study these undefined actin shaping proteins.
RESULTS
500bp 500bp
400bp 400bp
300bp 300bp
Figure 2: Expression of abp12 was confirmed in the zebrafish hair cell by reverse transcription polymerase chain reaction (RT-
PCR). Confirmation of abp12 expression in hair cell cDNA (+) is seen with two sets of interexonic primers, with water used in
place of cDNA in negative controls (--). (b) Expression of Abp12 was also confirmed in the mouse hair cell by RT-PCR with one
set of interexonic primers. Amplification of the cDNA products was further confirmed by sequencing the PCR
REFERENCES
1 Furness DN, Mahendrasingham S, Ohashi M, Fettiplace R, and Hackney C. The Dimensions
and composition of stereociliary rootlets in mammalian cochlear hair cells: comparison
between high- and low- frequency cells and evidence for a connection to the lateral
membrane. 2008; 28 (25): 6342-53
2 DeRosier DJ, Tilney LG. The structure of the cuticular plate, an in vivo actin gel. J Cell Biol.
1989; 109 (6Pt1): 2853-67
3 McDermott BM, Baucom JM, Hudspeth AJ. Analysis and functional evaluation of the hair- cell
transcriptome. Proceedings of the National Academy of Sciences. 2007; 104(28):11820-5.
4 McDermott BM, Asai Y, Baucom JM, Jari SD, Castellanos Y, et al. Transgenic labeling of hair
cells in the Zebrafish acousticolateris system. Gene Expr. Patterns. 2010;10:113-8
ACKNOWLEDGMENTS
We would like to thank members of Dr. Brian McDermott’s group for training and support. This
research was supported by Basil O'Connor Starter Scholar Research Award Grant No. 5-
FY07-663 from the March of Dimes Foundation, the Center for Clinical Research and
Technology at University Hospitals Case Medical Center, and National Institutes of Health (NIH)
Grant DC009437 and NIH Training Grants GM-08613 and GM008056-28.
Zebrafish hair cell
cDNA
Mouse hair
cell cDNA
+ --
a) b)
DISCUSSION:
• Actin-binding protein 12 is expressed in hair cells of both the mouse and zebrafish
• Immunolabeling of mouse hair cells and expression of GFP-tagged Abp12 in the zebrafish
show that Abp12 protein localizes to the hair cell cuticular plate in both species
• The Abp12 protein has been previously shown to have F-actin bundling and crosslinking
activity in other cell types.
• Thus, we hypothesize that Abp12 may be involved in crosslinking the actin filaments within
the cuticular plate. It is also possible that abp12 may function in connecting the actin
filaments of the stereociliary rootlets to the surrounding actin mesh of the cuticular plate.
• Future studies of Abp12 in the hair cell may help us to better understand the role of the
cuticular plate in hair cell morphology and function.
FUTURE DIRECTIONS:
1) Is Abp12 necessary for proper hair cell morphology and/ or hearing?
• We are generating zebrafish knockouts of abp12 using transcription activator-like
effector nucleases (TALENs)
2) If Abp12 is necessary for proper cuticular plate formation - is the cuticular plate
necessary for formation and/or maintenance of the hair bundle structure?
• Analysis of hair bundle morphology in abp12 knockout fish
3) Which domains of the Abp12 protein are necessary for its localization and function in
the cuticular plate
• Structure-function studies
pEGFP ABP-12
Not 1 Site
(added)
Xma 1 Site
(Added)
PCR
pEGFP-ABP12
5KB 1.5KB
Xma1 Xma1 Not1
TOPO PCR8TAVector
pEGFP ABP-12
PCR8 pEGFP
ABP12
TOPOVector
PV3B
Not1 Xma1
Mini-tol2
MT-SV-PVVector
Mini-tol2
Restriction Diges-
tion w/ Xma1 and
Not1
+T4 DNA Ligase
Ligation
PE- GFP ABP-12
PV3B MT-SV-PV
PEGFP-
ABP12
Vector
pEGFP- ABP12
Vector 1
Xma1 site
Purifying pEGFP- ABP12 Fragment
through Gel Digestion
3 GelVolumes Buffer QG
and 1 GelVolume Isopropanol
Extracted Gell
containing pEGFP-
ABP12 Fragment
MicroCentrifuge
Tube
Followed by standard Miniprep Purification Procedure
Followed byT4 DNA Ligase Ligation in which the
pEGFP- ABP12 Fragment was encorporated into the
MT- SV-PVVector containing the PV3B Promoter
METHODS:
RT-PCR: Expression of abp12 was confirmed in the hair
cell by reverse transcription polymerase chain reaction (RT-PCR)
using cDNA from purified zebrafish1 and mouse hair cells.
Amplifications were performed using interexonic primer pairs and
PCR parameters designed to amplify the cDNA product, but not
the genomic locus.
Immunofluorescence: Vestibular tissues were dissected from
6-12 month-old FVB/NJ mice, fixed in -20˚C methanol, and
incubated with anti-ABP12 primary antibody. Tissue was then
stained with Alexa 488 secondary antibody and Alexa 568-
phalloidin to label F-actin, and viewed by confocal microscopy.
Zebrafish transgenesis: Abp12 was cloned into a zebrafish
expression vector containing the hair cell parvalbumin 3b
promoter4 and GFP. Zebrafish embryos were injected with this
DNA construct at the 1-cell stage, and screened for GFP
expression at 4-6 days postfertilization (dpf). Somatic transgenic
fish were fixed in 4% paraformaldehyde, labeled with
fluorescent-coupled phalloidin, and viewed by confocal
microscopy.
Figure 3: Confocal images of whole-mount preparations of the
adult mouse saccule show abp12 immunofluorescence (green) at
the cuticular plate. F-actin of the hair bundle and cuticular plate
was labeled with fluorescent-coupled phalloidin (red). A lateral view
of a single hair cell is displayed at left, and a dorsal view of a group
of hair cells is displayed on the right with the fonticulus regions (the
kinocilium insertion point in the cuticular plate, which is free of
actin) of two hair cells indicated by the white arrowheads.
Figure 4: Confocal images of somatic transgenic zebrafish hair cells at 4dpf expressing
GFP-Abp12. F-actin of the hair bundle and cuticular plate is labeled with fluorescent-coupled
phalloidin (red). GFP-Abp12 (green) localizes primarily to the cuticular plate. A lateral view of
anterior macula hair cells is seen on the left, with hair bundles of two transgenic hair cells
indicated by the white arrows. A dorsal view of the cuticular plate region of posterior crista
hair cells is seen on the right, with the fonticulus of a transgenic hair cell indicated by the
white arrowhead.
ABSTRACT
The senses of hearing and balance depend on hair cells of the inner ear. Stereocilia
within the hair cell convert sound stimuli into electrical impulses that are interpreted by the
brain. Stereocilia are actin-filled rods held in place by an actin-filled meshwork called the
cuticular plate at the apical surface of the cell; this structure is termed the hair bundle. An
experiment was designed to identify key actin-shaping proteins of the stereocilia and
cuticular plate utilizing the zebrafish Danio Rerio as an effective model. Actin-Binding Protein
12 (ABP12) was identified as a likely candidate. A vector containing ABP12 cDNA, green
fluorescent protein cDNA (a reporter gene), and the hair cell promoter parvalbumin3b was
created from the original pEGFP-ABP12 vector through a series of restriction digestions and
ligations. PacI and NotI restriction sites were added and the entire product incorporated into
the PCR8 TOPO-TA vector. The MT/SV/PV zebrafish hair cell expression vector was then
linearized, then ligated with the pEGFP-ABP12 insert to produce the final vector. This
functional vector was injected into zebrafish embryos at the one cell stage to confirm
ABP12’s subcellular location in the cuticular plate. Though preliminarily results remain
inconclusive, ABP12 has been localized to the hair cell and cuticular plate. The localization
pattern of ABP12 in the hair cell will help to better understand ABP12’s potential role in the
hair cell and hearing.
In the zebrafish (Danio rerio) model, the middle and
outer ear are not present. In the inner ear, there are two
maculae and three cristae. The maculae (lined with an
intermixture of hair cells and supporting cells) are responsible
for detecting hearing and linear accelerations. Each macula is
attached to an otolith (calcium carbonate structure) by the
otolithic membrane. Three semicircular canals (fluid- filled
toroidal channels) that contain crista, which house hair cells,
are used to detect angular accelerations. Hair cells are also
present on the surface of fish in the lateral-line system. These
hair cells sense water movement.
The cuticular plate is an actin meshwork at the base of
the stereocilia. The cuticular plate is thought to act as a
substratum on which stereocilia pivot (DeRosier andTilney,
1989) and is held firmly in the soma by columns of
microtubules oriented parallel to the hair cell’s longitudinal axis
(Jaeger et al., 1994). Cables of filamentous actin (F-actin) run
along the length of the stereocilia mostly terminating near the
taper region (Floack and Cheung, 1977); however, a handful of
strands, which serve to anchor stereocilia, extend past the
tapered region to terminate within the cuticular plate actin
matrix (DeRosier and TIlney, 1989; Kitajiri et al., 2010).
+ - + - + -
The construction of the MT/SV/PV pEGFP- ABP12
vector containing the Zebrafish parvalbumin 3b promoter:
Primers introducing XmaI and NotI restriction sites to the left and
right bounds of the pEGFP and ABP12 fragments respectively were
used in a PCR reaction. The subsequent fragment (approx. 6.5kb)
was cloned into the TOPO PCR8 TA vector. A target MT/SV/PV
vector containing the parvalbumin 3b Zebrafish promoter (PV3B)
with NotI and XmaI sites was obtained. The PCR8 pEGFP- ABP12
vector was subsequently subcloned into the PV3B MT/SV/PV vector
in a two -step process. The PCR8 pEGFP- ABP12 vector was
digested with XmaI and NotI, and following a gel electrophoresis of
the product, the 1.5 kb ABP12 fragment and 5.0kb pEGFP fragment
were gel extracted respectively ligated (T4 ligase) into a digested
PV3B MT/SV/PV vector with open 5’ and 3’ at the open XmaI and
Not I sites. The final PV3B MT/SV/PV pEGFP ABP-12 vector was
then for consequent Zebrafish embryo injection.
The pattern is similar for all vertebrate species. The cuticular plate has substructure that
can be seen with electron microscopy, including linker proteins, which have not yet been
identified but are likely necessary to permit hearing. The meshwork of these structures
indicates that several actin- binding proteins are involved in the shaping of the cuticular
plate. We have identified candidate proteins that may localized to the hair cell’s actin-rich
structures by deep sequencing the hair-cell transcriptome. We here identify and describe
a promising candidate that may shape the cuticular plate of the hair cell, ABP12.
Promoters are stretches of DNA that initiate the transcription process of a
certain gene. Located upstream of the coding region of a gene. Promoters are the sites
at which RNA Polymerase I and II (for Eukaryotes- transcription of ribosomal RNA and
transcription of DNA respectively) bind to the DNA strand and begin transcription
downstream. The functional promoter is comprised of a Transcription Start Site (TSS),
binding sites transcription factions, and often a distal promoter further upstream.
Parvalbumin 3b gene is specifically expressed in hair cells.
We tested the hypothesis that ABP12 localizes to the hair bundle or the
cuticular plate. a suspected F- Actin shaping protein in the cuticular plate and stereocilia
In order to drive expression of GFP-ABP12 specifically in the hair cell, the MT/
SV/PV pEGFP- ABP12 vector was engineered to incorporate the parvalbumin 3b
promoter. For our experiments we use transient transgenisis in zebrafish.