UNDERSTANDING THE ROLE OF MACF1 IN THE HAIR CELL BY CONDITIONAL GENE
TARGETING IN MICE AND CHARACTERIZING THE LOCALIZATION PATTERN OF
DEMATIN
by
SHENYU SUN
Submitted in partial fulfillment of the requirements for the degree of
Master of Science
Thesis Advisor:
Brian M. McDermott Jr., Ph.D.
Department of Biology
CASE WESTERN RESERVE UNIVERSITY
August, 2018 CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Shenyu Sun
candidate for the degree of Master of Science degree*.
Committed Chair
Jessica Fox, Ph.D.
Committee Member
Brian M. McDermott, Ph.D.
Ruben Stepanyan, Ph.D.
Deborah Harris, M.S.
Date of Defense
06/05/2018
*We also certify that written approval has been obtained
for any proprietary material contained therein.
2 Table of Contents
Acknowledgments ...... 5
List of Tables ...... 6
List of Figures ...... 7
List of Abbreviations ...... 8
Abstract ...... 10
Chapter 1: Background Introduction ...... 11
1. Introduction ...... 12
1.1 Hearing in the vertebrates ...... 13
1.2 Stereocilia ...... 19
1.3 Cuticular plate ...... 20
1.4 Proteinaceous composition of the cuticular plate ...... 22
Chapter 2: Understanding the role of MACF1 in hair cell development by conditional gene targeting in mice ...... 27
2.1 Introduction ...... 28
2.1.1 Introduction of MACF1 ...... 28
2.1.2 Introduction to Cre-loxP system ...... 33
2.2 Materials and Methods ...... 35
2.2.1 Animals ...... 35
2.2.2 Genotyping ...... 35
2.3 Results ...... 38
2.4 Discussion ...... 44
Chapter 3: Identifying the expression pattern of Dematin, an actin-binding protein, in mouse inner ear ...... 46
3 3.1 Introduction ...... 47
3.2 Materials and Methods ...... 48
3.2.1 Animals ...... 48
3.2.2 Whole-mount immunolabeling ...... 48
3.2.3 Imaging ...... 49
4.3 Results ...... 49
4.4 Discussion ...... 52
References ...... 54
4 Acknowledgments
First and foremost, I would like to thank my advisor, Dr. Brian McDermott for his continuous support and direction. In the past three years, he encouraged me to become a professional biologist and created an exciting research environment in the lab to pursue the truth of science.
I would also like to thank the rest of my thesis committee: Dr. Jessica Fox, Dr.
Ruben Stepanyan and M.S. Deborah Harris for their valuable support and guidance during my graduation. I am also very thankful to Dr. Ruben Stepanyan and Dr.
Deborah Harris for their help with my experimental design and future career.
I am very grateful to all of the members of the McDermott lab, past and present.
Special thanks to Lana Pollock for her patient guidance in my first project. I would like to extend special thanks to Dr. Ahlam Salameh who helped me a lot in the Macf1 projects and Shaoyuan Zhu who taught me a lot in imaging. Thanks also to Caro
Fernando, Hoa Nguyen, Zongwei Chen, Shenxuan Wang, Haimeng Bai, Robin Woods
Davis, Kayla Kindig and Michael Dercoli for all their valuable advice and training.
I am also thankful to Dr. Kumar Alagramam, Dr. Suhasini Gopal, Daniel Chen, Dr.
Qing Yin Zheng and Dr. Martin Basch.for all of their help and for being great lab neighbors over the three years.
In the end, I especially thankful to my parents for their great love and support over the three years, forming the strongest backup force on my way pursuing the beauty of science.
5 List of Tables
Table 2.1 Primers used in Macf1 genotyping ...... 36
Table 2.2 PCR protocols used in Macf1 and Pax2-Cre genotyping ...... 37
Table 2.3 The Punnett square showing the predictdresults of the mouse genetic cross: (Female)
Macf flox/+ X Macf1+/-; Pax2-Cre+/- (Male) ...... 43
6 List of Figures
Figure 1.1 The components and structure of the human ear ...... 16
Figure 1.2 The structure of the human cochlea and the location of hearing hair cell ...... 17
Figure 1.3 The structure of a hair cell ...... 18
Figure 1.4 The types of cross-linking proteins in the cuticular plate ...... 24
Figure 1.5 Electron microscope images show the existence of cross-linking proteins in the
cuticular plate ...... 26
Figure 2.1 The structure of microtubule and actin cross-linking factor 1 (MACF1) ...... 30
Figure 2.2 The localization pattern of MACF1 in adult mouse vestibular hair cells ...... 31
Figure 2.3 The evidence of the linker bridging microtubule and actin in the cuticular plate ...... 32
Figure 2.4 The mechanism of Cre-loxP deletion ...... 34
Figure 2.5 The location and direction of loxP sites in gene Macf1...... 37
Figure 2.6 The major mouse genetic cross experiment to generate the Macf1flox/-; Pax2-Cre
conditional knockout mic ...... 40
Figure 2.7 Genotyping the Macf1flox and Pax2-Cre gene ...... 41
Figure 2.8 The electrophoresis gel genotyping results of Macf1 inner ear conditional knockout
mice ...... 42
Figure 3.1 Expression of dematin in mouse inner hair cells...... 50
Figure 3.2 Expression of dematin in mouse vestibular hair cells ...... 51
7 List of Abbreviations
ABR auditory brainstem response
ACF7 actin cross-linking family protein 7
BPAG1 bullous pemphigoid antigen 1
BSA bovine serum albumin cAMP adenosine monophosphate
CCV clathrin-coated vesicles
CH domain calponin-homology domain
CNS central nervous system
CP cuticular plate
DST dystonin
EDTA ethylenediaminetetraacetic acid
EPSCs excitatory postsynaptic currents
FCHSD1 FCH and double SH3 domains protein 1
G-actin globular-actin
GAR domain gas2-related domain
GOI gene of interest
GSR domain glycine-serine-arginine domain
HPD headpiece domain
IHCs inner hair cells
JAX The Jackson Laboratory
8 KO knockout cKO conditional knockout
MACF1 microtubule and actin cross-linking factor 1
MET mechanoelectrical transduction
OHCs outer hair cells
PBS phosphate buffered saline
PCR polymerase chain reaction
PFA paraformaldehyde
SNX9 sorting nexin 9
9 Abstract
Understanding the Role of MACF1 in the Hair Cell by Conditional Gene Targeting in
Mice and Characterizing the Localization Pattern of Dematin
Abstract
SHENYU SUN
The inner ear hair cells are mechanosensitive and significant for hearing. In the actin-based cuticular plate (CP) of hair cells, microtubule and actin cross-linking factor 1 (Macf1) has been hypothesized to form the linker bridging the microtubule and the actin. To further understand the role of Macf1 in hearing, knockout mice need to be obtained. As null mice of Macf1 is lethal at an early developmental stage, my work mainly focused on generating the conditional knockout of Macf1 via
Cre-loxP system in mouse inner ear. In addition, the actin-binding protein dematin is also reported to exist in the inner ear hair cells and be involved in regulating the actin cytoskeleton dynamics. However, the expression pattern of dematin remains unknown in the hair cells. I characterized the expression pattern of dematin in mouse inner ear hair cells, and this will help better understand the function of dematin in hair cells in the future.
10
Chapter 1: Background Introduction
11 1. Introduction
In vertebrates, hearing or auditory perception is the ability to recognize sound by detecting changes in the pressure, i.e., vibrations, of the animal’s surrounding medium. Affecting nearly one in 500 newborn babies, hearing loss is one of the most common congenital disabilities in the United States (Hilgert et al., 2009). Deafness in children significantly limits the global development and the functional ability of the child. These limitations compromise their quality of life and their social interactions with others(Hilgert et al., 2009).In Adults, age-related hearing loss imposes difficulties in verbal communication that disturb the regular daily activities (Dalton et al., 2003).
To better understand the factors that lead to hearing loss, it is crucial to comprehend the normal process of auditory perception. The human ear is composed of three major compartments: the outer ear, which includes the auricle, the external auditory meatus, and the tympanum (Figure 1.1). The middle ear, which consists of three linked bones or ossicles; the malleus, the incus, the stapes. These are contained in an air-filled middle ear cavity. The inner ear, which itself has two divisions: one for hearing, the cochlea, and the other for balance, the vestibular labyrinth.
12 1.1 Hearing in the vertebrates
When sound waves enter the external auditory meatus, it hits and vibrates the tympanum. The vibratory motions of the tympanum fluctuate the air pressure in the middle ear cavity and move the malleus. The physical movement of the malleus initiates mechanical vibrations that are then sent to the incus, which in turn communicates this mechanical energy with the innermost ossicle, the stapes. The ossicles then convey this mechanical energy to the cochlea at the oval window (not shown in the figure).
Three fluid-filled scalae or ducts; the scala vestibuli, scala media, and scala tympani, run the entire length of the cochlea (Kandel et al., 2012). The scala media is sandwiched between the other two compartments and contains the hearing apparatus, the organ of Corti, which is bathed in a high potassium fluid known as the endolymph. Specialize sensory cells known as hair cells are organized in a precise order within the organ of Corti. There are four rows of hair cells: one row forms the inner hair cells (IHCs) and three rows form the outer hair cells (OHCs). The general structure of both types of hair cells consists of the cylinder-like cell body and staircase-like stereocilia that project from the top of the cell body and bathe in the endolymph (Kandel et al., 2012). The organ of Corti lies on a membrane known as the basilar membrane. The stereocilia of the OHCs, but not the IHCs, contact another membrane known as the tectorial membrane (Kandel et al., 2012).
13 As the mechanical energy produced by the middle ear’s ossicles reaches the cochlea, it is transformed into hydraulic energy in the endolymph causing a wavy movement in the basilar membrane. During the basilar membrane movement, the
OHCs stereocilia stroke the tectorial membrane and deflect (Kandel et al., 2012)
(Figure 1.2). The deflection of OHCs stereocilia triggers the mechanoelectrical transduction (MET) channel at the tips of the bundles to open. The opening of the
MET channel enables the influx of Ca2+ and K+ into the cell. (Jaramillo and Hudspeth,
1991; Denk et al., 1995; Lumpkin and Hudspeth, 1995; Beurg et al., 2006). The deflection of the hair bundles toward the tallest stereocilium (Figure 1.3) opens the
MET channels and depolarize the hair cells, whereas the deflection of the bundles in the opposite direction closes the MET channels and hyperpolarizes the hair cells
(Kandel et al., 2012).
The depolarization of OHCs, causes the hair cells to contract. Reversely, the hyperpolarization of OHCs causes the cells to elongate. This contraction-elongation of the OHCs accentuates upward and downward movements of the basilar membrane. During the movement of the basilar membrane, the endolymph is forced to flow back and forth on the top of the free-floating stereocilia of IHCs. Comparable to the OHCs, IHCs depolarize when their bundles are deflected toward the tallest stereocilia and hyperpolarize when their bundles are deflected toward the opposite direction. The depolarization of the IHCs causes a release of glutamate from the presynaptic region at the base of the hair cell that initiates an action potential in the postsynaptic region in the afferent neuron producing excitatory postsynaptic currents
14 (EPSCs) (Glowatzki and Fuchs, 2002; Kandel et al., 2012). Thus, the sound wave transfers from mechanical signals into chemical signals, and finally into neuronal signals that are then conveyed to the central nervous system (CNS) for processing.
The death or the degeneration of cochlear hair cells that occurs due to genetic mutations, loud noise exposure, uptake of ototoxic medications, or otic infections, is considered one of the major factors that causes hearing loss (Izumikawa et al., 2005).
Hence, it is of great significance to understand the structure and the function of each part of the hair cells to help develop treatments for hearing impaired individuals. In this thesis, I will focus on two major structures of the auditory hair cells, the stereocilia and the cuticular plate (CP).
15
Figure 1.1 The components and structure of the human ear
The sound waves enter the external auditory meatus, vibrating the tympanum. These vibrations are delivered to the middle ear by three bones: the malleus, the incus, and the stapes. The stimuli finally reach the cochlea via the connection between the middle ear and the inner ear. Figure cited from Kandel et al., 2012. Used with the permission from the publisher.
16 A B
Figure 1.2 The structure of the human cochlea and the location of hearing hair cell
A) The structure of the human cochlea. The cochlea is composed of three major liquid-filled scalae or ducts: the scala vestibule, the scala media, and the scala tympani. The scala vestibule is near the oval window, whereas the scala tympani is near the round window. The scala media is an endolymph-filled tube where the
16,000 hair cells lie on the basilar membrane. B) The location of auditory hair cell.
The organ of Corti, lying on the basilar membrane, includes four rows of hair cells: one row of inner hair cells and three rows of outer hair cells. The tallest parts of all the outer hair cell bundles are connected to the tectorial membrane; however, the inner hair cells do not attach to the tectorial membrane. Those hair cells are
17 supported by different kinds of supporting cells, including the pillar cells and the
Deiters's cells. Figures cited from Kandel et al., 2012. Used with the permission from the publisher.
Figure 1.3 The structure of a hair cell
The hair cell contains actin-based structures: the cuticular plate (CP) and the hair bundles. Those bundles are arranged in a staircase-like shape. In mice, the kinocilium always degenerates at the time of birth in the hair cells in the organ of Corti, but remains in the vestibular hair cells. Deflecting the hair bundles' top from left to right will depolarize the hair cells, whereas deflection in the opposite direction 18 hyperpolarizes the hair cell. Figure cited from Kandel et al., 2012. Used with the permission from the publisher.
1.2 Stereocilia
Stereocilia are mechano-sensing organelles in the cochlear and vestibular hair cells. The stereocilia consist of paralleled actin-based bundles, which are the core structures in the inner ear hair cells and play a significant role in the mechanotransduction process during hearing (Flock and Cheung, 1977; Tilney et al.,
1980; Slepecky and Chamberlain, 1985; Bartles, 2000). In the stereocilia, fimbrin and espin link actin filaments together (Sekerkova et al., 2006). All stereocilia taper at the base and connect with the surface of the cuticular plate with densely-packed rootlet, which enables the stereocilia to deflect rigidly in response to vibration (Kimura, 1975;
Itoh, 1982; DeRosier and Tilney, 1989). One identified protein involved in the components of the rootlet is TRIOBP, is a cytoskeleton-associated protein whose mutation causes the human hereditary deafness DFNB28 (Kitajiri et al., 2010).
The stereocilia are connected to each other via several kinds of connections, which allows them to deflect and function simultaneously during the mechanotransduction process (Kandel et al., 2012). There are four major types of linkers between stereocilia: ankle links, shaft connectors, horizontal top connectors and tip links. The ankle links connect stereocilia at the base; the shaft connectors combine stereocilia near the central position of the bundles; the horizontal top
19 connectors link stereocilia on a level higher than the shaft; the tip links connect the apical surface of the shorter stereocilium to its taller neighbor's shaft (Vollrath et al.,
2007). Among all connections, the tip links are most important in the mechanotransduction process. As triggered via the deflection of stereocilia, the tension in the tip links forces the MET channel to open and Ca2+ and K+ to rush into the cell, which depolarizes the cell and leads to the release of synaptic vesicles
(Kandel et al., 2012). In this way, the stereocilia achieve the goal to transduce the mechanical waves into neuronal signals.
1.3 Cuticular plate
The CP is a hemisphere-like actin-based structure that locates at the apical part of the hair cell, forming the foundation of the actin-based hair bundles. The dense actin-based matrix of the CP prevents other cellular organelles from moving inside the area beneath the stereocilia (Corwin and Warchol, 1991; Kachar et al., 1997).
Hence, the CP presumably contributes to the regulation of the molecular composition of the apical plasma membrane, including the distribution of proteins and lipids. Therefore it allows the stereocilia to become an exclusive compartment
(Kachar et al., 1997).
The CP also plays a vital role in the mechanotransduction process. The cuticular plate does not directly connect to the belt desmosome via linkage of cytoskeleton structures (Figure 1.3 and Figure 1.5); it is hypothesized that the deflection of the
20 hair bundles leads to the movement of the CP and the continuous movements cause the adaptation process of mechanotransduction (Kachar et al., 1997). It was shown in the guinea pig vestibular hair cells that a 45: deflection of the stereocilia leads the
CP to tilt towards a direction opposite to the deflection, which could be an adjusting mechanism that the hair cells use to avoid the mechanical damage caused by over-deflection (Valat et al., 1991).
Studying the structural composition of the CP revealed the existence of several types of linkers inside the CP or between the CP and its adjacent cellular structures
(Figure 1.4, Figure 1.5). The actin filaments connect with each other via linking different proteins inside both the CP and the rootlets (Figure 1.4, Figure 1.5). There are also cross-linking proteins that combine both the CP actin filaments and the rootlet actin filaments. In addition, the CP actin filaments connect to the plasma membrane via another set of linking proteins and the microtubule inserting into the cuticular plate also cross-links the CP actin filaments by different kinds of linkage proteins. Thus far, the molecular components of all those linkers mentioned above have not been identified. Considering the fact that those linkages have a diverse function in the CP, it is possible that there could be different proteins contributing to the formation of each linker and each linker may also be constituted via multiple proteins.
21 1.4 Proteinaceous composition of the cuticular plate
To further understand the structure of CP, it is essential to study the protein composition of the CP. In addition to the α-actin (forming actin filaments), both
β-actin and γ-actin also exist in the CP; γ-actin is expressed in the overall CP, whereas
β-actin is mostly located at the rootlets (Furness et al., 2005; Perrin et al., 2010).
α-2-Spectrin (fodrin) is a protein that is expressed in the CP and has an actin-binding ability. Additionally, α-2-Spectrin may have the function to cross-link between actin filaments in a Ca2+-dependent manner. It also presumably connects the actin filaments to the plasma membrane (Scarfone et al., 1988). In previous work, it was shown that α-2-Spectrin plays a role in regulating the rigidity of the CP
(Scarfone et al., 1988; Demeˆmes and Scarfone, 1992; Vranceanu et al., 2012).
Plastin-1 (fimbrin) is also an actin-binding protein, which localizes to both the stereocilia and the CP (Slepecky and Chamberlain, 1985). Plastin-1 bundles the actin filaments in the stereocilia, but its function in the CP remains unknown.
Tropomyosin and α-actinin can interact with actin filaments and contribute to regulating the dynamics of the actin cytoskeleton (Lazarides, 1976). Tropomyosin locates at the rootlets inserting into the CP but is not expressed in the hair bundles above the rootlets (Slepecky and Chamberlain, 1985). Tropomyosin's function is considered to help stabilize the rootlet actin filaments. α-actinin mostly expresses at the apical part of the CP and also in the intervals between rootlets. The function of
α-actinin is also believed to cross-link actin filaments and regulate the dynamics of
22 the actin cytoskeleton in the CP (Wagner et al., 1999).
Myosin VI and Myosin VIIa are also significant protein constituents in the CP.
Myosin VI mostly expresses in the CP of both rodent and bullfrog hair cells (Hasson et al., 1997); it does not express in the rodent stereocilia but exists in bullfrog stereocilia. The myosin VI is thought to be involved in adequate intake of multiple epithelial receptors or transporters in the plasma membrane and have the function in the internalization of clathrin-coated vesicles (CCV) at the base of the hair bundles
(Pollard and Goldman, 2017). Myosin VIIa locates in both the stereocilia and the CP.
The mutation of myosin VIIa leads to the disorganization of stereocilia and cause the formation of abnormal bulges and the vesiculated area in the CP (Self et al., 1998). It remains unknown of myosin VIIa's function in the CP. But in the stereocilia, Myosin
VIIa is thought to anchor and hold the membrane, and connect the membrane to the actin cytoskeleton in the stereocilia (Kros et al., 2002). In addition, the harmonin b, one kind of actin-bundling protein, directly interacts with Myosin VIIa, also localizes both in the stereocilia and the boundary of the CP (Boëda et al., 2002), which may help to understand myosin VIIa's role in the CP.
As the formation and the polymerization of actin filaments are significant in the dynamics of the actin cytoskeleton, multiple proteins contribute to the regulation of the polymerization process in the CP. Profilin is a globular-actin (G-actin) monomer binding protein, which can prevent the G-actin from being involved in actin filament polymerization (Carlsson et al., 1977). It was reported that profilin expresses in the
CP (Slepecky and Ulfendahl, 1992). The FCH and double SH3 domains protein 1
23 (FCHSD1) and Sorting Nexin 9 (SNX9) are an interacting partner. They both co-localize in the CP, and both of them are considered to manipulate the polymerization of actin filaments (Cao et al., 2013). The interaction of both molecules would mostly enhance the activity of actin filament polymerization.
Figure 1.4 The types of cross-linking proteins in the cuticular plate
The CP and the stereocilia are both in gray, the actin filaments (green) and the microtubule (light blue) are also shown in the figure. (1) The actin filaments in the CP and the rootlets are linked by non-actin proteins (red). (2) The actin filaments inside the CP are combined with another set of non-actin proteins (purple). (3) Different linking proteins (orange) form the connection between the actin filaments which 24 belong to the rootlets. (4) The microtubule and the actin filaments inside the CP are cross-linked together by another linking protein (dark blue). (5) The actin filaments also connect to the surrounding plasma membrane via the linkage of other linking proteins (yellow). Figure cited from Pollock et al., 2015. Used with the permission from the publisher.
25 Figure 1.5 Electron microscope images show the existence of cross-linking proteins in the cuticular plate
A) An electron microscope image of the cuticular plate in a chicken hair cell. The arrowheads show the location of three rootlets and the arrows show the non-actin filaments which connect the rootlet actin filaments with each other or connect the rootlets actin filaments with other actin filaments that form the cuticular plate. Scale bar 0.1 µm. Cited from Hirokawa and Tilney, 1982. B) An electron microscope image of the connecting part between the cuticular and the plasma membrane in a chicken vestibule hair cell. The arrows show the connections of the cuticular plate actin filaments and the plasma membrane. Scale bar 0.1 µm. Cited from Hirokawa, 1986. C)
An electron microscope image of a guinea pig cochlear hair cell. The arrows show the linkage between the rootlet and the cuticular plate actin filaments. Cited from Arima et al., 1987. D) An electron microscope image with the process of three-dimensional tomographic reconstruction. A microtubule (mint) inserts into the cuticular plate (CP) of a zebrafish hair cell. The linkers (red) are shown to cross-link both the microtubule and the cuticular plate actin (pink). Scale bar 200 nm. Cited from Antonellis et al.,
2014. Whole figures cited from Pollock et al., 2015. Used with the permission from the publisher.
26
Chapter 2: Understanding the role of MACF1 in hair cell development by conditional gene targeting in mice
27 2.1 Introduction
2.1.1 Introduction of MACF1
Microtubule and actin cross-linking factor 1 (MACF1) is also called as actin control factor 7 (ACF7), α-trabeculin (Sun et al., 1999), and macrophin (Okuda et al.,
1999). MACF1 belongs to the spectraplakin family identified in vertebrates and the other member in the spectraplakin family is called as bullous pemphigoid antigen 1
(BPAG1), also known as dystonin (DST) (Leung et al., 2002; Jefferson et al., 2004;
Sonnenberg and Liem 2007). MACF1 possesses both actin-binding and microtubule-binding abilities. The CH1 and CH2 domains can bind to actin, located at the N-terminus, and the GAR and GSR domains at the C-terminus have the capacities to bind microtubules (Figure 2.1). Those structures allow MACF1 to become a factor to bridge both actin filaments and microtubule. The plakin domain and the sprctrin repeat domain mainly contribute structurally and are also the signature domains of the spectraplakin family.
Up to now, substantial work has been made to study the function of MACF1 in non-auditory cells. The mutation of Macf1 is lethal to mice at the gastrulation stage
(Chen et al., 2006). Cultured mice Macf1-/- fibroblasts can still be alive, but microtubules in those cells will no longer grow near the actin-rich cortical regions, which is consistent with its role as a microtubule and actin cross-linking factor
(Kodama et al., 2003). In the epidermis, the conditional knockout of MACF1 disturbs the microtubules in targeting the focal adhesions along actin filaments and results in
28 the impairment of epidermal migration (Wu et al., 2011). The specific knockout of
Macf1 in the nervous system also causes the malfunction of neuronal migration in the process of moving through the cortical plate of the brain (Goryunov et al. 2010;
Ka et al. 2014). As shown above, a feasible way to study the function of MACF1 is based on the conditional knockout in the specific tissue.
In the recent study of MACF1 in hearing hair cells, our lab has characterized the localization pattern of MACF1 (Figure 2.2) and found the evidence of linker proteins bridging microtubule and actin in the cuticular plate (Figure 2.3). Because MACF1 has the ability to bind both actin and microtubule, we could hypothesize that MACF1 could be the potential linker protein bridging the microtubule and the actin. As mice that lack MACF1 die at an early developmental stage, a feasible way to test this hypothesis is to generate conditional knockout mice of Macf1 in the inner ear, and this project is one of the primary tasks in my research.
29
Figure 2.1 The structure of microtubule and actin cross-linking factor 1
(MACF1)
Here is shown the major components of MACF1. The N-terminus of MACF1 includes
CH1 and CH2 domains, and those two domains can bind to actin. The plakin domain can function to interact with junctional proteins and other molecules in the cytoplasm. The spectrin repeats can make the protein more flexible. Those two
EF-hands could bind calcium. The GAR domain and GSR domain form the microtubule-binding domain at the C-terminus. CH domain, calponin-homology domain; GAR domain, gas2-related domain; GSR domain, glycine-serine-arginine domain. Figure cited from Antonellis et al., 2014. Used with the permission from the publisher.
30
Figure 2.2 The localization pattern of MACF1 in adult mouse vestibular hair cells
Mouse vestibular hair cells that are immunostained with the MACF1 antibody (green), phalloidin (red) and tubulin antibody (blue). Phalloidin specifically labels actin. A)
MACF1 localizes between actin (red) and microtubules (blue). B) The licalization pattern of MACF1. MACF1 expresses at the boundary of the cuticular plate and forms a circle along the fonticulus. Figures cited from Antonellis et al., 2014. Used with the permission from the publisher.
31
Figure 2.3 The evidence of the linker bridging microtubule and actin in the cuticular plate
The evidence of linker proteins (red) bridging actin matrix (pink) and microtubule
(mint). The boundary of actin matrix shows in blue; mitochondria are shown in yellow and green. A) 2D transmission electron microscope image of a zebrafish hair cell shows a microtubule inserting in the cuticular plate (CP). Asterisk is the apical surface of the hair cell. B) A 1 nm-thick slice 3D reconstruction of figure A. C) An amplified view of the rectangular region in figure B. Asterisk shows the filamentous linkage between microtubule and actin. D) Manual segmentation and surface rendering of the densities of interest through 3D reconstruction. E) Amplified region of interest in figure D. Scale bar, 200 nm. Figures cited from Antonellis et al., 2014.
Used with the permission from the publisher.
32 2.1.2 Introduction to Cre-loxP system
The Cre-loxP conditional knockout system was generated initially based on the
Cre enzyme discovered in the P1 bacteriophage (Cox et al., 2012). The function of Cre enzyme in P1 bacteriophage is to help the replication and the development of new virus (Soper, 2014). Cre can specifically target a 34-bp short DNA sequence called loxP site, which does not exist in the mouse genome and has directionality (Cox et al.,
2012). The Cre recombinase can recombine genes flanked by loxP sites (floxed sequence) and lead to different recombination results depending on the location and direction of the loxP sites, including inversion (two loxP sites on the same allele and in the opposite direction ) , deletion (two loxP sites on the same allele and in the same direction, Figure 2.4), or translocation (two loxP sites on different allele and in the same direction) of DNA (Cox et al., 2012; Soper, 2014; Nagy, 2000). In this way, if there is a promoter added to the Cre-loxP system to drive the tissue specific expression of Cre, it will be able to function as a tissue-specific knockout, this technology has already been utilized in the study of the inner ear and dozens of promoters have previously been reported to work well in the conditional knockout
(cKO) technique based on Cre-loxP system (Cox et al., 2012).
33
Figure 2.4 The mechanism of Cre-loxP deletion
The mechanism of using the Cre-loxP system to knock out the gene of interest (GOI).
The Cre recombinase encoded by Cre gene will target the floxed gene and recombine those two loxP sites to knock out the target gene. It needs to be noted that the loxP sites have directionality, as shown above, both sites in the same direction will lead to deletion. If both loxP sites are in the opposite direction, it will cause inversion. And if both loxP sites are in the same direction but on different alleles, it will result in translocation. Figure cited from Soper, 2014. Reproduced with permission of The
Jackson Laboratory.
34 2.2 Materials and Methods
2.2.1 Animals
Macf1 flox mice (Wu et al., 2011) were generously donated by Dr. Elaine Fuchs from The Rockefeller University. Pax2-Cre mice were got from the Mutant Mouse
Resource & Research Center (stock #10569). FVB/NJ (wild-type) mice were obtained from The Jackson Laboratory (JAX).
2.2.2 Genotyping
The mouse ear-punch tissues were obtained following standard procedures. The tissues were then added into 50 µl of base solution (25 mM NaOH, 0.2 mM Na2EDTA, pH 12) and incubated at 95 °C for 30 minutes to extract DNA. After incubation, 50 µl of neutralization solution (40 mM Tris-HCl, pH 5) was added into the extraction solution, followed by centrifugation at 3000 rpm for 5 minutes at room temperature.
The DNA extraction samples were restored at -20 °C .
The target sequence in DNA extraction samples was amplified by polymerase chain reaction (PCR). The PCR reaction mix includes: 13.375 µl of DNAse-free water,
0.5 µl of dNTPs, 0.5 µl of 10 µM forward primer, 0.5 µl of 10 µM reverse primer,
0.125 µl of Taq DNA polymerase (New England BioLabs Inc.), 2.5 µl of 10x standard buffer (New England BioLabs Inc.) and 5 µl of DNA extraction template. The primer information and the PCR protocols were shown in Table 2.1 and Table 2.2. The Macf1 flox forward and reverse primer target sites were shown in Figure 2.5.
35 The genotyping results were presented via agarose electrophoresis gel results.
1 % agarose gel and 1 Kb plus ladder (Invitrogen) were used in this process.
Table 2.1 Primers used in Macf1 genotyping
Allele Primers Expected band sizes
Macf1flox 10697 Macf1flox F 5'-AAAGAAACGGAAATACTGGCC-3' 700 bp floxed allele band
10698 Macf1flox R 5'-GCAGCTTAATTCTGCCAAATTC-3' 660 bp wild-type allele band
Pax2-Cre Pax2-Cre_F 5'-GCCTGCATTACCGGTCGATGCAACGA-3' Around 700 bp band
Pax2-Cre_R 5'-GTGGCAGATGGCGCGGCAACACCATT-3'
Recombined Rec_F 5'-AAAGAAACGGAAATACTGGCC-3' 1000 bp (recombined band)
Macf1 Rec_R 5'-AAGTTCAGTCCGGGTCATCG-3' 3000 bp (non-recombined band)
Cntrl_F 5'-GGTCACCCGACTGCTAGATG-3' 879 bp (positive control band)
Cntrl_R 5'-ATGCTGCCTGATCCAGGGAATGAG-3'
36
Table 2.2 PCR protocols used in Macf1 and Pax2-Cre genotyping
Allele Denaturation Annealing Elongation Round
Macf1flox 95°C /30s 56°C /30s 68°C /1min 35
Pax2-Cre 94°C /45s 67°C /45s 72°C /1min 35
Recombined Macf1 95°C /30s 55°C /30s 72°C /2.5min 35
Figure 2.5 The location and direction of loxP sites in gene Macf1
In mouse gene Macf1, one loxP site locates between exon 10 and exon 11; the other
loxP locates between exon 13 and exon 14 as the red triangles show. Once the
recombination occurs, the exon 11, 12 and 13 will be deleted, and the primers shown
in purple arrows will not be able to amplify the sequence. Figure cited from Pollock
LM., 2016. Used with the permission from the publisher.
37 2.3 Results
This project mainly focused on the generation of mice, which have the conditional knockout of Macf1 in the inner ear. This conditional knockout generation was based on the Cre-loxP system. I used the Pax2 promoter to guide the expression of Cre recombinase specifically in the inner ear, and thus Macf1 should only be knocked out in the cells belonging to the inner ear (Figure 2.5).
Among the abundant genetic crosses involved in this project, the essential genetic cross was Macf1flox/+ x Macf1+/-; Pax2-Cre+/- (Figure 2.6). This cross was the final genetic step to generate the conditional knockout mice, whose genotype was
Macf1flox/-; Pax2-Cre+/-. Several other genetic crosses were used to maintain the strains of the parental mouse lines for this major genetic cross or other strains that may indirectly contribute to generating the conditional knock out (cKO) mice. The maintained strains of mice included: Macf1flox/+;Pax2-Cre+/-, Macf1flox/+, Macf1flox/-,
Pax2-Cre+/- and FVB/NJ. To genotype all the mice mentioned above, the electrophoresis gel results of PCR products were a good way to achieve this goal.
Different alleles were represented by different bands (Figure 2.7). These gel results were easy and swift to identify the genotype of numerous mice, including Macf1flox/-;
Pax2-Cre+/-, Macf1flox/+; Pax2-Cre+/-, Macf1flox/+, Macf1flox/-, and Pax2-Cre+/-. The minus
(-) allele was identified by another two pairs of primers (not shown).
The potential mice genotype distribution of the major genetic cross is shown in the Punnett square (Table 2.3), and the conditional knockout mice, whose theoretical
38 probability among the pups is 1/8. However, at the very beginning, the ratio was meager and was just around 1 out of 50. The first identified conditional knockout mouse was mouse 23 C10 (Figure 2.8), which came from the genetic cross: (Female)
Macf1flox/+ x Macf1+/-; Pax2-Cre+/- (Male). In the past, the relation between mouse gender with Pax2-Cre+/- was not considered, both Macf1+/-; Pax2-Cre+/- and
Macf1flox/+; Pax2-Cre+/- were also used to generate cKO mice equally. Dr. Andy Groves
(who generated the Pax2-Cre mice strain (Ohyama and Groves, 2004)) told us not to use the female mouse to carry Pax2-Cre+/-, as his group found that Pax2-Cre was expressed everywhere in the female ovary which would lead to the death of all cKO mice. Another empirical rule was generated from hundreds of genotyping results that in the genetic cross, when Pax2-Cre+/- was with Macf1+/- in mice, it had higher chance to generate the cKO mice. From the major genetic cross: (Female) Macf1flox/+ x Macf1+/-; Pax2-Cre+/- (Male), 7 cKO mice had already been generated, however, no cKO mice were identified from the cross: (Female) Macf1flox/+; Pax2-Cre+/- x Macf1+/-
(Male). This indicates the sex of the parental mice were important for generation of target mice.
In this way, the major genetic cross was finally concluded as :
(Female) Macf1flox/+ x Macf1+/-; Pax2-Cre+/- (Male)
According to recent genotyping results, the ratio of the cKO mice in the total pups from this major genetic cross is also close to the theoretical probability 1 out of
8.
39
Figure 2.6 The major mouse genetic cross experiment to generate the
Macf1flox/-; Pax2-Cre conditional knockout mice
This shows the major genetic cross to get the conditional knockout mice. In the cross,
Cre is downstream of the Pax2 promoter. It was reported that Pax2-Cre expresses in the otic placode at E8.5 and then in the cochlea, vestibular organs, spiral and vestibular ganglia at P0 (Ohyama and Groves, 2004). In the cross, Macf1flox/+ x
Macf1+/-; Pax2-Cre, the probability of generating the conditional knockout mice
Macf1flox/-; Pax2-Cre is 12.5%. Theoretically, those mice should have mutant cells in their inner ear, but the rest of the mice remains normal. Reproduced with permission of The Jackson Laboratory. 40
Figure 2.7 Genotyping the Macf1flox and Pax2-Cre gene
The electrophoresis gel results show the bands representing different alleles. Each lines represent the same mouse in both figures. A) PCR amplified bands representing
Macf1flox gene. The upper band is the flox band; the lower band stands for the wild-type (+) band. Mouse 1 genotype: Macf1flox/-; mouse 2-4 genotype: Macf1flox/+; mouse 5 genotype: Macf1+/+ B) PCR amplified bands representing Pax2-Cre gene. The band is the Pax2-Cre band, whose genotype is Pax2-Cre+/- (mouse 1 and 2);
Pax2-Cre-/- shows a negative result in the figure (mouse 3 and 4).
41
Figure 2.8 The electrophoresis gel genotyping results of Macf1 inner ear conditional knockout mice
The electrophoresis gel results show the genotype of the Macf1flox/-; Pax2-Cre conditional knockout mouse. A) The red box shows the genotyping result for Macf1 gene of the mouse numbered 23C10. Considering the possible results of the genetic cross: Macf1+/- x Macf1flox/+; Pax2-Cre, the genotyping result of the 23C10 mouse in
Macf1 gene is Macf1flox/-. B) The red box shows the genotyping result for the
Pax2-Cre gene of the mouse numbered 23C10. The genotyping result of the 23C10 mouse in Pax2-Cre gene is Pax2-Cre+/-.
42
Table 2.3 The Punnett square showing the predicted results of the mouse genetic cross: (Female) Macf flox/+ X Macf1+/-; Pax2-Cre+/- (Male)
43 2.4 Discussion
The goal of this project is to generate the cKO mice of Macf1 in the inner ear.
We have generated several possible cKO mice. Verifying the cKO and characterizing the phenotype will be the next step. To figure out whether cKO of Macf1 will cause hearing defects in mice, together with Dr. Ahlam Salameh, we are now characterizing the cKO mouse hearing capacity via auditory brainstem response (ABR) test. To verify the cKO of Macf1 in the inner ear hair cells, we will also image the inner ear hair cells by immunostaining using MACF1 (ACF7) antibodies to compare the expression pattern of MACF1 between the wild-type and cKO inner ear hair cells. We can also characterize the defects caused by the cKO at the cellular level by imaging the morphological changes of different categories of hair cells. Another potential step could be the ultrastructural analysis, which can take images at the location where microtubules insert into the CP to see if the linker protein still exist. Another potential phenotype could be that the microtubules do not extend to the CP region as the result of the MACF1 deletion.
As the microtubule and actin filaments are tracts for transport in the cell, the linker proteins that guide and shape them may be important for this process. In the cell, the microtubule is much more like the highway, and the meshed actin filaments are similar to the local roads (Pollard et al., 2017). Along microtubules and actin filaments different transport proteins move; these transfer the cargo to each other at the junctions between two types of cytoskeletons (Pollard et al., 2017). In this way,
44 the linker protein may play a much more essential role in the cargo transferring process between two kinds of transporters at junctions of different cytoskeleton fibers. The linker proteins may contribute to changing the conformation of one transport protein so that it can release the cargo and then help the other transport protein on a different type of fiber to have higher affinity with the cargo and proceed the following transport (Pollard et al., 2017). Perhaps, the linker protein can also help recruit the other proteins to change the conformation of the transport proteins to achieve this goal instead of doing this job by themselves.
45
Chapter 3: Identifying the localization pattern of Dematin, an actin-binding protein, in mouse inner ear
46 3.1 Introduction
Dematin is a type of actin-binding protein, which consists of a headpiece domain (HPD) at its C-terminus; this region is present in all members of the villin family proteins. The rest of dematin is a combination of gelsolin-repeats. HPD is vital for dematin's actin-binding property and the phosphorylation of the HPD caused by adenosine monophosphate (cAMP)- dependent protein kinase will negatively regulate the actin-binding ability of dematin in vitro (Husain-Chishti et al., 1988;
Siegel and Branton, 1985). It was reported that in erythrocytes, dematin functions as an anchor for the junctional complex and it could directly bind to actin, adducin
(Azim et al., 1995) and spectrin, which shows its role in anchoring these proteins to the plasma membrane (Lu et al., 2015). However, the mechanism underlying this process remains unknown.
In the mouse inner ear, the expression pattern of dematin has not been fully characterized. In zebra fish hair cells, Our lab showed localization of dematin in both zebra fish stereocilia and cuticular plates (Ince, 2015). As the stereocilia are actin-based bundles and play an essential role in the mechanotransduction process in hearing, characterizing the localization pattern of all proteins of the stereocilia and the cuticular plate are vital to understand these important structures.
47 3.2 Materials and Methods
3.2.1 Animals
Wild-type FVB/NJ mice were obtained from The Jackson Laboratory (JAX).
3.2.2 Whole-mount immunolabeling
Adult mice (over one-month-old) were euthanized following the standard protocols. Mice were decapitated, and the bony cochlea was dissected out. These cochleae were fixed in 4% paraformaldehyde (PFA) at 4 °C overnight. If the cochlea were for the dissection of the organ of Corti, the fixed cochlea would be decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for an extra two days at 4°C. The vestibule or organ of Corti dissected from the cochlea were washed in phosphate buffered saline (PBS) for 30 minutes, permeabilized in 0.05% Triton X-100 for two hours at room temperature, and then were blocked in blocking solution (2% bovine serum albumin (BSA) with 0.05% Triton X-100) for 2 hours at room temperature. The tissues were then incubated with anti-dematin mouse primary antibody overnight
(around 16 hours) at 4°C. The anti-dematin mouse primary antibody was diluted
1:500 in blocking solution. Then, the tissues were washed 3 times for 10 minutes each in blocking solution and incubated in Alexa 488 anti-rabbit secondary antibody
(Invitrogen) at a 1:200 dilution and Alexa 633 phalloidin at a 1:40 dilution in blocking solution for 2 hours. The tissues were washed 3 times for 10 minutes each in PBS and mounted in medium Prolong Diamond (Thermo Fisher).
48 3.2.3 Imaging
The tissues were imaged using a Leica SP8 confocal microscope, 63x or 100x objective lens, with the processing of original images by deconvolution software.
4.3 Results
In this project, I identified the localization pattern of dematin in mouse inner ear hair cells via immunostaining and imaging.
In mouse inner hair cells, dematin is seen to localize in both the stereocilia and the cuticular plate of the inner hair cells (Figure 3.1). Moreover, a large section of the protein concentrates on the lower halves of the hair bundles. However, in this figure, no expression is shown in the outer hair cells, neither in the cuticular plate (Figure
3.1 B) nor in the stereocilia (Figure 3.1 C).
In the vestibular hair cells, the expression pattern of dematin is much more clear.
Almost all the vestibular hair cells have the dematin expression in both the cuticular plate and the hair bundles (Figure 3.2). Similar to the inner hair cells, the expression concentrates to the lower half part of the hair bundles (Figure 3.2 A) and it shows that dematin encircles the cuticular plate boundary (Figure 3.2 C) in the vestibular hair cells. It also needs to be noticed that the expression of dematin shows weaker expression in a section of the vestibular hair cells, which remains unknown what caused this phenomenon (Figure 3.2 A).
49
Figure 3.1 Expression of dematin in mouse inner hair cells
Dematin localizes in mouse inner hair cells. dematin (green), actin (red) A) Dematin expresses in mouse inner hair cells. B, C) Dematin localizess both in the stereocilia and the cuticular plate of inner hair cells. B) The lower stack of figure A. C) The upper stack of figure A. D) Dematin's localization pattern of high resolution in the IHCs.
50
Figure 3.2 Localization of dematin in mouse vestibular hair cells
Dematin is expressed in mouse vestibular hair cells. Dematin (green), actin (red). A) dematin localizes in both the stereocilia and the cuticular plates in mouse vestibular hair cells. The red line shows the boundary of hair cells in different expression level.
B) Actin-based stereocilia. C) The mixed image of both figure A and B. The white arrows show that dematin encircles the cuticular plate. D) Dematin's localization pattern of high resolution in the vestibular hair cells.
51 4.4 Discussion
The expression pattern of dematin in the mouse inner ear was identified in this project. Via immunostaining and imaging, dematin is shown to express in inner hair cells and vestibular hair cells. In both inner and vestibular hair cells, dematin localizes to the cuticular plate and the stereocilia. In the stereocilia, it mostly localizes at the lower halves. In the vestibular hair cells, dematin encircles the cuticular plate boundary and form a loop (Figure 3.2 C).
In addition to characterizing the expression pattern, generating the KO mice that lack dematin could be another efficient method to understand the function of dematin in the hair cells. As dematin is an actin-binding protein and it shows that it expresses in both the hair bundles and the cuticular plates, the deletion of dematin could cause defects in the morphologies of hair bundles or impair the hearing process of mice. Another way to characterize dematin would be to find the proteins, which could interact with dematin and see if those proteins could be able to function in the hearing process. Co-immunostaining of those interacting proteins with dematin could also be an effective way to characterize the function of dematin.
Dematin is reported to function in the erythrocytes and help anchor the actin-spectrin cytoskeleton to the plasma membrane (Lu et al., 2015). This protein could have a similar role in inner ear hair cells. It could be one component of the complexes bridging the actin-based hair bundles with the plasma membrane and maintain the rigid structure of stereocilia. However, the exact function of dematin
52 remains unknown in the hair cells.
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