Liquid-liquid phase separation in hair cell stereocilia development and maintenance

Abstract

As an emerging concept, liquid-liquid phase separation (LLPS) in biological systems has shed light on the formation mechanisms of membrane-less compartments in cells. The process is driven by multivalent interactions of biomolecules such as proteins and/or nucleic acids, allowing them to form condensed structures. In the inner ear hair cells, LLPS-based biomolecular condensate assembly plays a vital role in the development and maintenance of stereocilia, the mechanosensing organelles located at the apical surface of hair cells. This review aims to summarize recent findings on the molecular basis governing the LLPS of Usher syndrome-related gene-encoding proteins and their binding partners, which may ultimately result in the formation of upper tip-link density and tip complex density in hair cell stereocilia, offering a better understanding of this severe inherited disease that causes deaf-blindness.

Graphical Abstract

1. Introduction

Cell compartmentalization allows the segregation of distinct components in a confined space; therefore, various biochemical reactions can be spatiotemporally well-controlled. Compartmentalization is usually facilitated by physical separation using lipid membranes. For example, the endoplasmic reticulum (ER) and mitochondria are surrounded by lipid membranes. However, many compartments lack membranes, such as centrosomes [1], [2] and stress granules [3] in the cytoplasm and nucleoli [4] and Cajal bodies [5], [6] in the nucleus. In the last decade or two, it has been recognized that many of these membrane-less compartments are formed by a physicochemical process called liquid-liquid phase separation (LLPS). Although well-studied in polymer/material science, LLPS has not captured biologists’ attention until recently [7], [8], [9]. LLPS in biological systems refers to the process in which macromolecules, when above threshold concentrations, form a condensed phase autonomously and are separated from the aqueous cellular environment. It is suggested that multivalent interactions, mediated by folded domains or intrinsically disordered regions of proteins, as well as nucleic acids and chromatin, thermodynamically drive LLPS [10], [11], [12], [13]. LLPS-mediated condensate formation has been implicated in a variety of biological processes, including cellular homeostasis [3], [14], [15], transcriptional and translational regulation [16], [17], [18], [19], synaptic transmission in neurons [20], [21], [22], [23], [24], [25], immune signaling [26], [27], [28], [29], and mechanoelectrical transduction (MET) in hair cells [30], [31], [32], which is the focus of this review.

Hearing, or auditory perception, enables us to explore the outer world and communicate with one another. This perception of sound is facilitated by our ears. Our ears can convert mechanical vibrations (i.e., sound waves) into electrical signals (neural signals), which can be interpreted by our brains [33], [34]. Anatomically, the human ear consists of three parts: the outer, middle, and inner ear. While the vestibular system is essential for balance, the cochlea in the inner ear is dedicated to hearing. Within the cochlea, the organ of Corti is where sound is detected and converted [35]. In mammals, the organ of Corti is comprised of ∼15,000–30,000 mechanosensory hair cells, which are positioned on a thin basilar membrane and grouped into two subtypes: three rows of outer hair cells and one row of inner hair cells (Fig. 1A) [36]. On top of each hair cell lies a bundle of hair-like actin-rich structures called stereocilia, arranged in a staircase pattern [37], [38] (Fig. 1B). These stereocilia serve as mechanical sensors, which can undergo sound-induced deflection. Deflection can trigger the opening of the MET channels located at the tip of the stereocilia, leading to the depolarization of the hair cell [39].

Fig. 1.

Schematic of the organ of Corti and hair cell for sound perception. (A) A diagram of the transverse section of the organ of Corti in the mammalian cochlea. (B) A schematic showing a hair cell with stereocilia lying at its apical surface in a staircase pattern. Important compartments including the tip-link, upper tip-link density (UTLD), lower tip-link density, mechanoelectrical transduction (MET) machinery, tip complex, ankle link complex, and ribbon synapse are highlighted. The transient kinocilium is also shown with a dashed contour.

Not surprisingly, developmental defects or damage to this sophisticated structure can lead to hearing loss [40], [41], [42], [43]. According to a World Health Organization (WHO) report in 2021, it is estimated that more than 1.5 billion people experience some degree of hearing loss. Genetic background, health conditions, lifestyles, and environmental factors can contribute to the onset of hearing loss. For newborns with deafness, genetic factors account for over 50%. Hereditary hearing loss can be classified into two main groups, non-syndromic and syndromic hearing loss [44]. Non-syndromic hearing loss can be categorized into three types according to their chromosomal locations and the patterns of inheritance. DFNA represents autosomal dominant deafness; DFNB stands for autosomal recessive deafness; and DFNX denotes X-linked deafness. There are currently 11 known syndromes associated with hearing loss, including Usher syndrome and Alport syndrome. Usher syndrome is one of the best-known forms. Genetic studies have identified several genes (USH genes) linked with this disease [45]. The proteins encoded by these genes and their interacting partners play essential roles in this elaborate sound perception process, especially in terms of stereocilia development, maintenance, and mechanoelectrical transduction of hair cells [46], [47], [48], [49]. This review will summarize some of our recent findings and those of others on complex assembly mediated by USH gene-encoding proteins.

2. Usher syndrome and USH proteins

Usher syndrome is an autosomal recessive disorder characterized by both deafness and blindness. The frequency has been estimated as roughly 4–17 per 100,000 people. According to the severity of the symptoms, Usher syndrome can be subdivided into type I (USH1), type II (USH2), and type III (USH3). Children with USH1 are severely deaf at birth and have vision problems at a young age. USH2 patients have congenital moderate-to-severe deafness and retinitis pigmentosa in adolescence. USH1 and USH2 account for about 95% of all cases. People with USH3 are not born deaf but often develop hearing loss and/or night blindness in adolescence. The currently known USH genes that might cause this disease include six (USH1B [50], [51], USH1C [52], [53], USH1D [54], [55], [56], USH1F [57], [58], USH1G [59] and USH1J [60]) for USH1, three (USH2A [61], USH2C [62] and USH2D [63]) for USH2, and one (USH3A [64]) for USH3.

Among these proteins, the cadherin proteins CDH23 (Cadherin-23, encoded by USH1D) and PCDH15 (Protocadherin-15, encoded by USH1F) form the tip-link through head-to-head interaction via their extracellular cadherin (EC) repeats and connect the tip of one stereocilium to the adjacent taller one [65], [66] (Fig. 1B). The tip-link has been proposed to convey sound-induced force to the MET channel located at the tip of the shorter stereocilium. At either end of the cytoplasmic side of the tip-link, an electron-dense plaque was observed under the electron microscope, named the upper tip-link density (UTLD) and lower tip-link density (LTLD), respectively [67]. The UTLD contains three USH1 proteins, a molecular motor Myo7a (unconventional myosin VIIa, encoded by USH1B), a PDZ (PSD95/Dlg/ZO1) domain-containing protein Harmonin (encoded by USH1C), and an ankyrin repeats (ANK) and SAM (sterile α motif) domain-containing protein Sans (encoded by USH1G) (Fig. 1B). These interact with one another to form a tripartite complex and finally anchor to the cytoplasmic tail (CT) of CDH23 [68], [69], [70], [71], [72], [73]. The LTLD is thought to associate with the MET channel and its auxiliary subunits. PCDH15 at the LTLD side binds to the MET machinery components LHFPL5 (lipoma HMGIC fusion partner-like 5, also known as TMHS), TMIE (transmembrane inner ear), and the putative channel TMC1 (Transmembrane channel-like protein 1) [74], [75], [76], [77]. In addition, another USH1 protein CIB2 (calcium and integrin binding family member 2, encoded by USH1J) was reported as an auxiliary subunit that interacted with the cytoplasmic loop of TMC1 (Fig. 1B) [78], [79]. The three USH2 gene-encoding proteins Whirlin (encoded by USH2D), Usherin (encoded by USH2A), and Adgrv1 (adhesion G protein-coupled receptor v1; also called very large G-protein coupled receptor, vlgr1; encoded by USH2C) are better known as components of the ankle link complex (Fig. 1B) [80], [81], [82]. The ankle link is located at the basal region of stereocilia [83]. In mammals, the ankle link transiently appears during developmental stages and disappears prior to the onset of hearing [84]. The adhesion molecule Usherin interacts with Adgrv1 to form the ankle link. Their CTs contain a PBM (PDZ binding motif), which can bind to the PDZ domain-containing protein Whirlin and PDZD7 (PDZ domain-containing protein 7, encoded by PDZD7, a modifier of Usher syndrome) [85], [86]. Clarin-1 (encoded by USH3A) is the only known USH3 protein. It is a tetraspanin-like membrane protein. It has been proposed to function as a key component of hair cell stereocilia and ribbon synapse [87], [88]. In ribbon synapse, it can interact with the presynaptic calcium channel CaV1.3 and the scaffold protein Harmonin (Fig. 1B) [89].

3. Molecular mechanisms governing UTLD formation

Tip-link densities have been noticed for over 30 years [90]. Head movements or sound-induced vibration incessantly generate stretching forces to the stereocilia. The strength of the forces was estimated to be > 100 pN [91], which is strong enough to pull the single-pass transmembrane proteins CDH23 or PCDH15 out of the membrane. Intuitively, the protein-containing dense plagues can serve as the soil to bury the roots (CTs) of the tip-link to sustain such large stretching forces. Recent studies have proposed a possible mechanism for how the densities are formed and how the tip-links are rooted in the densities [30].

As mentioned above, three USH1 proteins, Harmonin, Sans, and Myo7a, build up the central part of the UTLD (Fig. 2A) [72], [73]. Both Harmonin and Sans are scaffold proteins. Harmonin has multiple isoforms and can be grouped into three classes, a, b, and c [53]. Harmonin-b is restrictedly expressed in the cochlea during embryonic and early postnatal stages, whereas the a and c isoforms are widely expressed in several tissues and throughout life. Harmonin-a contains an N-terminal HHD (Harmonin homology domain) domain, three PDZ domains, a putative coiled-coil region (CC1) between the second and third PDZ domains, and a C-terminal PBM; Harmonin-b contains an additional predicted coiled-coil region (CC2) and a proline-serine-threonine-rich (PST) region between the CC1 and the third PDZ domain; and Harmonin-c lacks the third PDZ domain. Sans consists of four N-terminal ankyrin repeats, an unstructured central region (CEN), and a C-terminal SAM domain followed by a PBM. Myo7a contains a motor domain at the head, five IQ motifs and a single α helix (SAH) at the neck, and a pair of MyTH4 (myosin tail homology 4)-FERM (band 4.1, Ezrin, Radixin, Moesin) tandems separated by a SH3 (Src homology 3) domain at the tail. These three proteins interact with each other to form a tripartite complex (Fig. 2B).

Fig. 2.

Molecular basis underlying formation of the upper tip-link density. (A) A schematic of the inner ear hair cell stereocilia showing the tip-link and the electron-dense plaques at both ends. (B) Schematics showing the domain organizations and the interaction network of the core protein components in the upper tip-link density. EC, extracellular cadherin repeat; NBM, N-terminal domain binding motif; PBM, PDZ binding motif; HHD, Harmonin homology domain; PDZ, PSD-95/discs-large/ZO-1; CC, coiled-coil; PST, proline-serine-threonine rich; ANK, ankyrin repeat; CEN, central domain; SAM, sterile α motif; SAH, single α-helix; MyTH4, myosin tail homology 4; FERM, protein 4.1/ezrin/radixin/moesin; SH3, Src homology 3. (C–F) Ribbon diagram representations of the reported overall structures of several binary complexes: Harmonin/Sans (C) (PDB: 3K1R), Myo7a/Sans (D) (PDB: 3PVL), Myo7a/Harmonin (E) (PDB: 5MV9), and Harmonin/CDH23 (F) (PDB: 2KBR and 2KBS).

Detailed biochemical and structural studies have revealed the molecular basis governing tripartite complex formation. Harmonin binds Sans tightly, with a dissociation constant (Kd) of a few nM [71]. The Harmonin HHD and PDZ1 couple together and form a stable integral supramodule. The Sans PBM inserts into the pocket of the Harmonin PDZ1 and the Sans SAM domain extensively contacts the Harmonin PDZ1 to generate such a strong affinity (Fig. 2C). Sans can also interact with Myo7a with a high affinity (Kd of ∼50 nM) [92]. The interaction is mediated by the CEN region of Sans and the N-terminal MyTH4-FERM tandem domain (NMF). The N-terminal half of CEN binds the central inter-lobe interfaces of the cloverleaf-shaped FERM domain and the C-terminal half binds the positively charged surface of the MyTH4 domain (Fig. 2D). These two tight binary interactions are sufficient for tripartite complex formation. Interestingly, later studies showed that Harmonin PDZ3-PBM together could bind the C-terminal MyTH4-FERM tandem domain (CMF), though with relatively low affinity (Fig. 2E) [93], [94].

The multivalent interaction among Harmonin/Sans/Myo7a is reminiscent of postsynaptic density (PSD) assemblies [20], which can drive LLPS. Similarly, both PSD and UTLD are electron-dense structures. Indeed, our previous study demonstrated that these three proteins together were sufficient to form a condensed structure when overexpressed in cells [30]. The condensates are liquid-like, can fuse, and are highly dynamic, which are hallmarks of LLPS. Moreover, the formation of condensates depends on each binary interaction. Usher syndrome-related point mutations that affect the binary interactions can increase the concentration threshold required for LLPS formation and thus weaken the ability to form condensates. Other factors might also contribute to LLPS. The cochlea-specific exon 68 of CDH23 encodes a sequence capable of mediating the dimerization of CDH23 CT and thus can form large protein assemblies with Harmonin [95]. Sans also tends to self-associate via its SAM domain, a domain well-known for its ability to form oligomers. Moreover, F-actin, the major cytoskeleton of stereocilia, provides a series of binding sites for the motor domain of Myo7a. All these factors can further increase the valency and thus promote LLPS.

Anchoring of UTLD to the tip-link depends on the interaction between Harmonin and CDH23, the tip-link component at the UTLD side (Fig. 2B). Each Harmonin molecule contains two binding sites for CDH23 [70]. The Harmonin HHD and the PDZ2 domain recognize an internal sequence and the PBM in CDH23 CT, respectively (Fig. 2F). LLPS clusters Harmonin at high concentrations (estimated as high as a few mM). Although the affinities of both interactions are moderate at the micromolar range, such a high concentration of Harmonin can significantly increase affinity for CDH23, providing anchorage to the tip-link to withstand ceaseless sound wave-induced stretching. Besides, our recent study implied that under such high concentrations, the CC1 of Harmonin tends to form an antiparallel dimer [96]. Dimer formation can create a new surface that is absent in its monomeric form. This newly formed surface could potentially interact with unknown proteins and recruit them to the condensates.

Intriguingly, brush border microvilli, another actin-based protrusion resembling stereocilia, also contain a highly homologous complex (called intermicrovillar adhesion complex, IMAC) consisting of Harmonin, ANKS4B, Myo7b, and the cadherin-related proteins CDHR2/CDHR5 [97], [98]. Similar multivalent interactions among Harmonin/ANKS4B/Myo7b also promote LLPS in vitro and in heterologous cells. Although no electron microscope-based evidence has shown the existence of electron-dense plaques in microvilli, we suggest that Harmonin/ANKS4B/Myo7b might also form tip-link density-like condensates in microvilli for the stable anchorage of the CTs of CDHR2 and CDHR5.

4. Row 1-specific tip complex condensate formation

A distinctive feature of hair cell stereocilia is their staircase-like pattern, which makes the tallest row (row 1) of stereocilia of particular interest. It is widely accepted that the staircase-like pattern of stereocilia is controlled by planar cell polarity mediated by kinocilia, which are specialized microtubule-based cilia found in hair cells [36], [99]. Each hair cell typically contains a single kinocilium. At the early stage of stereocilia development, the kinocilium moves from the center to the edge of the apical surface. Then, the stereocilia closest to the kinocilium elongate first, followed by the second and third rows, forming a staircase pattern. Attachment of the tallest stereocilia to the kinocilium relies on a protein-based link known as the kinociliary link (Fig. 3A). The CD2 isoform of the USH1 protein PCDH15 was reported as one of the components [100]. At the stereocilia side of the kinocilium link, a set of tip-locating proteins can form a complex (hereafter referred to as the tip complex) to determine the row identity and promote elongation of the stereocilia (Fig. 3A) [101], [102], [103]. Recent studies have shed light on the assembling mechanisms of the tip complex [31], [32].

Fig. 3.

Molecular basis underlying row-1-specific tip complex density formation. (A) Schematic of the inner ear hair cell row-1 stereocilia and kinocilium showing the tip complex density. (B) Schematics showing the domain organizations and interaction network of the core protein components in the tip complex density. TPR, tetratricopeptide repeat; GL, GoLoco motif; PR, proline-rich region; PTB, phosphotyrosine-binding domain; WBD, Whirlin binding domain; IDR, intrinsically disordered region. (C–E) Ribbon diagram representations of the solved structures of several binary complexes: Whirlin/Myo15a (C) (PDB: 6KZ1), GPSM2/Whirlin (D) (PDB: 7EP7), and Gαi/GPSM2 (E) (PDB: 4G5Q).

To date, at least five proteins (Whirlin, Myo15a, Eps8, GPSM2, and Gα_i) have been identified as core components of the tip complex (Fig. 3B). Whirlin is a USH2 protein and shares similar domain architecture to the USH1 protein Harmonin. It contains an N-terminal HHD domain, two PDZ domains, another HHD domain, a proline-rich (PR) region, and a third PDZ domain. Myo15a (unconventional myosin XVa) is an F-actin-based molecular motor. Analogous to Myo7a, it contains a motor domain, two IQ motifs, and two consecutive MyTH4-FERM tandems separated by a SH3 domain. It also contains a large N-terminal unstructured region proceeding the motor domain and an insertion at the first MyTH4 domain. Eps8 (epidermal growth factor receptor pathway substrate 8) is an F-actin capping protein that controls actin dynamics. It comprises an N-terminal PTB (phosphotyrosine-binding) domain, a middle SH3 domain, and a C-terminal actin-binding SAM domain. These three proteins are thought to be essential for stereocilia elongation. Mice carrying mutations in these genes (whirler, shaker-2, and Eps8-knockout mice) were profoundly deaf and showed shortened stereocilia [63], [102], [103], [104]. The remaining two are polarity-regulating proteins. GPSM2 (G-protein-signaling modulator 2, also called LGN) contains eight tetratricopeptide repeats (TPRs) at its N-terminus, an unstructured linker in the middle, and four GoLoco (GL) motifs at the C-terminus. It is known to regulate spindle orientation during asymmetric cell division [105]. Its GL motifs can bind the GDP-bound form of the α subunit of heterotrimeric G protein and serve as a GDI (guanine nucleotide dissociation inhibitor) [106]. Mutations of the Gpsm2 gene have been linked to a rare disease called Chudley-McCullough syndrome (CMS) [107], an autosomal recessive genetic disease comprising early-onset sensorineural hearing loss and other structural brain abnormalities. A recent study also reported that GPSM2 colocalized with Whirlin at the tips of row 1 stereocilia [108], [109].

Similar to the UTLD assemblies mentioned above, the tip complex is also formed by multivalent interactions. A well-characterized interaction is that between Whirlin and Myo15a, in which the PDZ3 domain binds the PBM of Myo15a using a typical PDZ-PBM binding mode (Fig. 3C) [31], [110]. The Whirlin HHD1 can bind Eps8. The region of Eps8 responsible for binding (WBD for Whirlin binding domain) is located between its PTB and SH3 domain [31], [102], which contains an α-helical domain as predicted by Alphafold2 [111]. Eps8 can also use its N-terminal PTB domain to interact with the C-terminal MyTH4-FERM of Myo15a. The self-association properties of Whirlin and Eps8 further contribute to the valency. Whirlin can also associate with GPSM2 by binding the inner groove of TPR with a short stretch of sequence in the PR region (Fig. 3D) [32], [108]. GPSM2 can further recruit Gα_i. Each GL motif binds Gα_i with a typical binding mode (Fig. 3E), generating a 1:4 stoichiometry [112], [113].

As expected, recent studies showed that Whirlin/Eps8/Myo15a together could undergo LLPS [31]. The above-mentioned multivalent interactions are the main driving forces. Whirlin can self-associate with itself and autonomously undergo LLPS under high concentrations, contributing to condensate formation as well. GPSM2 alone can also undergo LLPS, possibly via multivalent electrostatic interactions between the positively charged linker and other negatively charged surfaces [32]. The truncated mutant form of GPSM2 lacking both the linker and GL motifs found in CMS patients fails to form condensates. By binding Whirlin, GPSM2 can significantly promote LLPS of the Whirlin/Myo15a/Eps8 condensates. GPSM2 further recruits Gα_i to the condensates and forms the 5 × tip complex density (TCD). With the actin-bundling activity of the C-terminal SAM domain of Eps8, the 5 ×TCD, when compared to the 3 ×TCD, can spark bundling of F-actin much more robustly, providing a possible underlying mechanism for coordinating polarity cues and elongation of the tallest stereocilia [32].

5. Perspective

Over the last decade, LLPS in biological systems has gained significant attention. Our studies, along with others’, have revealed how LLPS contributes to the development and maintenance of hair cell stereocilia, the mechanosensing organelle important for hearing. Our earlier study proposed that the formation of UTLD via LLPS might strengthen anchoring of the tip-link and thus withstand the stretching force [30]. Zhu’s group showed that the building of TCD could enhance the actin-bundling activity and therefore promote the elongation of stereocilia [31], [32]. It is worth noting that LLPS may also play a role in other aspects of the auditory system.

The LTLD is likely formed via LLPS. Similar to the UTLD, the LTLD is the lower anchoring site for the tip-link and undergoes constant stretching. The LTLD might also cluster the MET channels. However, the molecular components of the LTLD are not clearly known. Myo15a and Eps8l2 (a homolog of Eps8), which localize at the tips of stereocilia and are necessary for stereocilia elongation, may be two potential components [101], [114], [115]. The CT of PCDH15 can bind Whirlin [116], but the presence of Whirlin in the LTLD is still under debate [117]. Another actin-bundling protein, Espin-1 or its paralog Espin-like, is likely transported to the tips of stereocilia by class III unconventional myosin Myo3a/Myo3b [118], [119], [120], [121]. Studies have also shown that Espin can interact with Whirlin [122]. It will be interesting to determine whether Espin is a potential component of LTLD. More information is needed to understand how the LTLD is assembled and how it is linked to the MET channels.

Ankle links are transiently formed during the stereociliary development stage. The two PDZ domain-containing proteins, Whirlin and PDZD7, are thought to be cytoplasmic scaffold proteins that anchor the ankle link components Usherin and Adgrv1 [81]. A dense web was observed under an electron microscope, indicating that the ankle link complex can also undergo LLPS [80], [84]. The earlier TCD study showed that Whirlin alone could form condensates in vitro and in cells. How the Whirlin condensates orchestrate the assembly of other ankle link components remains elusive. As the ankle link will disappear upon stereocilia maturation, the ankle link condensates should be dynamically regulated, differing from the UTLD/LTLD condensates.

CRediT authorship contribution statement

Jianchao Li: Writing – reviewing and editing.

Conflict of interest

The author declares no competing interest.