Control of stereocilia length during development of hair bundles.

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later-in early stage IV-and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J, and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction's role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.

The Atad5 RFC-like complex is the major unloader of proliferating cell nuclear antigen in Xenopus egg extracts.

Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC. However, RFC, Ctf18-RLC, and even some subcomplexes of their shared subunits are capable of unloading PCNA in vitro, leaving an ambiguity in the division of labor in eukaryotic clamp dynamics. By using a system that specifically detects PCNA unloading, we show here that Atad5-RLC, which accounts for only approximately 3% of RFC/RLCs, nevertheless provides the major PCNA unloading activity in Xenopus egg extracts. RFC and Ctf18-RLC each account for approximately 40% of RFC/RLCs, while immunodepletion of neither Rfc1 nor Ctf18 detectably affects the rate of PCNA unloading in our system. PCNA unloading is dependent on the ATP-binding motif of Atad5, independent of nicks on DNA and chromatin assembly, and inhibited effectively by PCNA-interacting peptides. These results support a model in which Atad5-RLC preferentially unloads DNA-bound PCNA molecules that are free from their interactors.

GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region.

GIPC3 has been implicated in auditory function. Here, we establish that GIPC3 is initially localized to the cytoplasm of inner and outer hair cells of the cochlea and then is increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at 1 month of age. Cuticular plates of Gipc3KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks and the cuticular plate. Several of immunoprecipitated proteins contained GIPC family consensus PDZ-binding motifs (PBMs), including MYO18A, which bound directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell junction proteins to shape the cuticular plate.

Mechanotransduction by hair cells: models, molecules, and mechanisms.

Mechanotransduction, the transformation of mechanical force into an electrical signal, allows living organisms to hear, register movement and gravity, detect touch, and sense changes in cell volume and shape. Hair cells in the inner ear are specialized mechanoreceptor cells that detect sound and head movement. The mechanotransduction machinery of hair cells is extraordinarily sensitive and responds to minute physical displacements on a submillisecond timescale. The recent discovery of several molecular constituents of the mechanotransduction machinery of hair cells provides a new framework for the interpretation of biophysical data and necessitates revision of prevailing models of mechanotransduction.

Loss of Baiap2l2 destabilizes the transducing stereocilia of cochlear hair cells and leads to deafness.

KEY POINTS: Mechanoelectrical transduction at auditory hair cells requires highly specialized stereociliary bundles that project from their apical surface, forming a characteristic graded 'staircase' structure. The morphogenesis and maintenance of these stereociliary bundles is a tightly regulated process requiring the involvement of several actin-binding proteins, many of which are still unidentified. We identify a new stereociliary protein, the I-BAR protein BAIAP2L2, which localizes to the tips of the shorter transducing stereocilia in both inner and outer hair cells (IHCs and OHCs). We find that Baiap2l2 deficient mice lose their second and third rows of stereocilia, their mechanoelectrical transducer current, and develop progressive hearing loss, becoming deaf by 8 months of age. We demonstrate that BAIAP2L2 localization to stereocilia tips is dependent on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is a new key protein required for the maintenance of the transducing stereocilia in mature cochlear hair cells. ABSTRACT: The transduction of sound waves into electrical signals depends upon mechanosensitive stereociliary bundles that project from the apical surface of hair cells within the cochlea. The height and width of these actin-based stereocilia is tightly regulated throughout life to establish and maintain their characteristic staircase-like structure, which is essential for normal mechanoelectrical transduction. Here, we show that BAIAP2L2, a member of the I-BAR protein family, is a newly identified hair bundle protein that is localized to the tips of the shorter rows of transducing stereocilia in mouse cochlear hair cells. BAIAP2L2 was detected by immunohistochemistry from postnatal day 2.5 (P2.5) throughout adulthood. In Baiap2l2 deficient mice, outer hair cells (OHCs), but not inner hair cells (IHCs), began to lose their third row of stereocilia and showed a reduction in the size of the mechanoelectrical transducer current from just after P9. Over the following post-hearing weeks, the ordered staircase structure of the bundle progressively deteriorates, such that, by 8 months of age, both OHCs and IHCs of Baiap2l2 deficient mice have lost most of the second and third rows of stereocilia and become deaf. We also found that BAIAP2L2 interacts with other key stereociliary proteins involved in normal hair bundle morphogenesis, such as CDC42, RAC1, EPS8 and ESPNL. Furthermore, we show that BAIAP2L2 localization to the stereocilia tips depends on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is key to maintenance of the normal actin structure of the transducing stereocilia in mature mouse cochlear hair cells.

A cryo-tomography-based volumetric model of the actin core of mouse vestibular hair cell stereocilia lacking plastin 1.

Electron cryo-tomography allows for high-resolution imaging of stereocilia in their native state. Because their actin filaments have a higher degree of order, we imaged stereocilia from mice lacking the actin crosslinker plastin 1 (PLS1). We found that while stereocilia actin filaments run 13 nm apart in parallel for long distances, there were gaps of significant size that were stochastically distributed throughout the actin core. Actin crosslinkers were distributed through the stereocilium, but did not occupy all possible binding sites. At stereocilia tips, protein density extended beyond actin filaments, especially on the side of the tip where a tip link is expected to anchor. Along the shaft, repeating density was observed that corresponds to actin-to-membrane connectors. In the taper region, most actin filaments terminated near the plasma membrane. The remaining filaments twisted together to make a tighter bundle than was present in the shaft region; the spacing between them decreased from 13 nm to 9 nm, and the apparent filament diameter decreased from 6.4 to 4.8 nm. Our models illustrate detailed features of distinct structural domains that are present within the stereocilium.

A comparative analysis of genetic hearing loss phenotypes in European/American and Japanese populations.

We present detailed comparative analyses to assess population-level differences in patterns of genetic deafness between European/American and Japanese cohorts with non-syndromic hearing loss. One thousand eighty-three audiometric test results (921 European/American and 162 Japanese) from members of 168 families (48 European/American and 120 Japanese) with non-syndromic hearing loss secondary to pathogenic variants in one of three genes (KCNQ4, TECTA, WFS1) were studied. Audioprofile characteristics, specific mutation types, and protein domains were considered in the comparative analyses. Our findings support differences in audioprofiles driven by both mutation type (non-truncating vs. truncating) and ethnic background. The former finding confirms data that ascribe a phenotypic consequence to different mutation types in KCNQ4; the latter finding suggests that there are ethnic-specific effects (genetic and/or environmental) that impact gene-specific audioprofiles for TECTA and WFS1. Identifying the drivers of ethnic differences will refine our understanding of phenotype-genotype relationships and the biology of hearing and deafness.

Stereocilia Rootlets: Actin-Based Structures That Are Essential for Structural Stability of the Hair Bundle.

Sensory hair cells of the inner ear rely on the hair bundle, a cluster of actin-filled stereocilia, to transduce auditory and vestibular stimuli into electrical impulses. Because they are long and thin projections, stereocilia are most prone to damage at the point where they insert into the hair cell's soma. Moreover, this is the site of stereocilia pivoting, the mechanical movement that induces transduction, which additionally weakens this area mechanically. To bolster this fragile area, hair cells construct a dense core called the rootlet at the base of each stereocilium, which extends down into the actin meshwork of the cuticular plate and firmly anchors the stereocilium. Rootlets are constructed with tightly packed actin filaments that extend from stereocilia actin filaments which are wrapped with TRIOBP; in addition, many other proteins contribute to the rootlet and its associated structures. Rootlets allow stereocilia to sustain innumerable deflections over their lifetimes and exemplify the unique manner in which sensory hair cells exploit actin and its associated proteins to carry out the function of mechanotransduction.

ELMOD1 Stimulates ARF6-GTP Hydrolysis to Stabilize Apical Structures in Developing Vestibular Hair Cells.

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ADP-ribosylation factor (ARF) small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMO domain-containing protein 1 (ELMOD1), a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we analyzed mice of both sexes from a strain lacking functional ELMOD1 [roundabout (rda)]. In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5; large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cell's apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles compared with controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle.SIGNIFICANCE STATEMENT Assembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia.

Electron cryo-tomography of vestibular hair-cell stereocilia.

High-resolution imaging of hair-cell stereocilia of the inner ear has contributed substantially to our understanding of auditory and vestibular function. To provide three-dimensional views of the structure of stereocilia cytoskeleton and membranes, we developed a method for rapidly freezing unfixed stereocilia on electron microscopy grids, which allowed subsequent 3D imaging by electron cryo-tomography. Structures of stereocilia tips, shafts, and tapers were revealed, demonstrating that the actin paracrystal was not perfectly ordered. This sample-preparation and imaging procedure will allow for examination of structural features of stereocilia in a near-native state.

A Model for Link Pruning to Establish Correctly Polarized and Oriented Tip Links in Hair Bundles.

Tip links are thought to gate the mechanically sensitive transduction channels of hair cells, but how they form during development and regeneration remains mysterious. In particular, it is unclear how tip links are strung between stereocilia so that they are oriented parallel to a single axis; why their polarity is uniform despite their constituent molecules' intrinsic asymmetry; and why only a single tip link is present at each tip-link position. We present here a series of simple rules that reasonably explain why these phenomena occur. In particular, our model relies on each of the two ends of the tip link having distinct Ca2+-dependent stability and being connected to different motor complexes. A simulation employing these rules allowed us to explore the parameter space for the model, demonstrating the importance of the feedback between transduction channels and angled links, links that are 60° off-axis with respect to mature tip links. We tested this key aspect of the model by examining angled links in chick cochlea hair cells. As implied by the assumptions used to generate the model, we found that angled links were stabilized if there was no tip link at the tip of the upper stereocilium, and appeared when transduction channels were blocked. The model thus plausibly explains how tip-link formation and pruning can occur.

Neuroplastin Isoform Np55 Is Expressed in the Stereocilia of Outer Hair Cells and Required for Normal Outer Hair Cell Function.

UNLABELLED: Neuroplastin (Nptn) is a member of the Ig superfamily and is expressed in two isoforms, Np55 and Np65. Np65 regulates synaptic transmission but the function of Np55 is unknown. In an N-ethyl-N-nitrosaurea mutagenesis screen, we have now generated a mouse line with an Nptn mutation that causes deafness. We show that Np55 is expressed in stereocilia of outer hair cells (OHCs) but not inner hair cells and affects interactions of stereocilia with the tectorial membrane. In vivo vibrometry demonstrates that cochlear amplification is absent in Nptn mutant mice, which is consistent with the failure of OHC stereocilia to maintain stable interactions with the tectorial membrane. Hair bundles show morphological defects as the mutant mice age and while mechanotransduction currents can be evoked in early postnatal hair cells, cochlea microphonics recordings indicate that mechanontransduction is affected as the mutant mice age. We thus conclude that differential splicing leads to functional diversification of Nptn, where Np55 is essential for OHC function, while Np65 is implicated in the regulation of synaptic function. SIGNIFICANCE STATEMENT: Amplification of input sound signals, which is needed for the auditory sense organ to detect sounds over a wide intensity range, depends on mechanical coupling of outer hair cells to the tectorial membrane. The current study shows that neuroplastin, a member of the Ig superfamily, which has previously been linked to the regulation of synaptic plasticity, is critical to maintain a stable mechanical link of outer hair cells with the tectorial membrane. In vivo recordings demonstrate that neuroplastin is essential for sound amplification and that mutation in neuroplastin leads to auditory impairment in mice.

Tip-link protein protocadherin 15 interacts with transmembrane channel-like proteins TMC1 and TMC2.

The tip link protein protocadherin 15 (PCDH15) is a central component of the mechanotransduction complex in auditory and vestibular hair cells. PCDH15 is hypothesized to relay external forces to the mechanically gated channel located near its cytoplasmic C terminus. How PCDH15 is coupled to the transduction machinery is not clear. Using a membrane-based two-hybrid screen to identify proteins that bind to PCDH15, we detected an interaction between zebrafish Pcdh15a and an N-terminal fragment of transmembrane channel-like 2a (Tmc2a). Tmc2a is an ortholog of mammalian TMC2, which along with TMC1 has been implicated in mechanotransduction in mammalian hair cells. Using the above-mentioned two-hybrid assay, we found that zebrafish Tmc1 and Tmc2a can interact with the CD1 or CD3 cytoplasmic domain isoforms of Pcdh15a, and this interaction depends on the common region shared between the two Pcdh15 isoforms. Moreover, an interaction between mouse PCDH15-CD3 and TMC1 or TMC2 was observed in both yeast two-hybrid assays and coimmunoprecipitation experiments. To determine whether the Pcdh15-Tmc interaction is relevant to mechanotransduction in vivo, we overexpressed N-terminal fragments of Tmc2a in zebrafish hair cells. Overexpression of the Tmc2a N terminus results in mislocalization of Pcdh15a within hair bundles, together with a significant decrease in mechanosensitive responses, suggesting that a Pcdh15a-Tmc complex is critical for mechanotransduction. Together, these results identify an evolutionarily conserved association between the fish and mouse orthologs of PCDH15 and TMC1 and TMC2, supporting the notion that TMCs are key components of the transduction complex in hair cells.

A short splice form of Xin-actin binding repeat containing 2 (XIRP2) lacking the Xin repeats is required for maintenance of stereocilia morphology and hearing function.

Approximately one-third of known deafness genes encode proteins located in the hair bundle, the sensory hair cell's mechanoreceptive organelle. In previous studies, we used mass spectrometry to characterize the hair bundle's proteome, resulting in the discovery of novel bundle proteins. One such protein is Xin-actin binding repeat containing 2 (XIRP2), an actin-cross-linking protein previously reported to be specifically expressed in striated muscle. Because mutations in other actin-cross-linkers result in hearing loss, we investigated the role of XIRP2 in hearing function. In the inner ear, XIRP2 is specifically expressed in hair cells, colocalizing with actin-rich structures in bundles, the underlying cuticular plate, and the circumferential actin belt. Analysis using peptide mass spectrometry revealed that the bundle harbors a previously uncharacterized XIRP2 splice variant, suggesting XIRP2's role in the hair cell differs significantly from that reported in myocytes. To determine the role of XIRP2 in hearing, we applied clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome-editing technology to induce targeted mutations into the mouse Xirp2 gene, resulting in the elimination of XIRP2 protein expression in the inner ear. Functional analysis of hearing in the resulting Xirp2-null mice revealed high-frequency hearing loss, and ultrastructural scanning electron microscopy analyses of hair cells demonstrated stereocilia degeneration in these mice. We thus conclude that XIRP2 is required for long-term maintenance of hair cell stereocilia, and that its dysfunction causes hearing loss in the mouse.

Molecular remodeling of tip links underlies mechanosensory regeneration in auditory hair cells.

Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism's life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca(2+)-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.

Correlation of actin crosslinker and capper expression levels with stereocilia growth phases.

During development of the chick cochlea, actin crosslinkers and barbed-end cappers presumably influence growth and remodeling of the actin paracrystal of hair cell stereocilia. We used mass spectrometry to identify and quantify major actin-associated proteins of the cochlear sensory epithelium from E14 to E21, when stereocilia widen and lengthen. Tight actin crosslinkers (i.e. fascins, plastins, and espin) are expressed dynamically during cochlear epithelium development between E7 and E21, with FSCN2 replacing FSCN1 and plastins remaining low in abundance. Capping protein, a barbed-end actin capper, is located at stereocilia tips; it is abundant during growth phase II, when stereocilia have ceased elongating and are increasing in diameter. Capping protein levels then decline during growth phase III, when stereocilia reinitiate barbed-end elongation. Although actin crosslinkers are readily detected by electron microscopy in developing chick cochlea stereocilia, quantitative mass spectrometry of stereocilia isolated from E21 chick cochlea indicated that tight crosslinkers are present there in stoichiometric ratios relative to actin that are much lower than their ratios for vestibular stereocilia. These results demonstrate the value of quantitation of global protein expression in chick cochlea during stereocilia development.

Distinct energy metabolism of auditory and vestibular sensory epithelia revealed by quantitative mass spectrometry using MS2 intensity.

Measuring the abundance of many proteins over a broad dynamic range requires accurate quantitation. We show empirically that, in MS experiments, relative quantitation using summed dissociation-product ion-current intensities is accurate, albeit variable from protein to protein, and outperforms spectral counting. By applying intensities to quantify proteins in two complex but related tissues, chick auditory and vestibular sensory epithelia, we find that glycolytic enzymes are enriched threefold in auditory epithelia, whereas enzymes responsible for oxidative phosphorylation are increased at least fourfold in vestibular epithelia. This striking difference in relative use of the two ATP-production pathways likely reflects the isolation of the auditory epithelium from its blood supply, necessary to prevent heartbeat-induced mechanical disruptions. The global view of protein expression afforded by label-free quantitation with a wide dynamic range reveals molecular specialization at a tissue or cellular level.

The local forces acting on the mechanotransduction channel in hair cell stereocilia.

In hair cells, mechanotransduction channels are located in the membrane of stereocilia tips, where the base of the tip link is attached. The tip-link force determines the system of other forces in the immediate channel environment, which change the channel open probability. This system of forces includes components that are out of plane and in plane relative to the membrane; the magnitude and direction of these components depend on the channel environment and arrangement. Using a computational model, we obtained the major forces involved as functions of the force applied via the tip link at the center of the membrane. We simulated factors related to channels and the membrane, including finite-sized channels located centrally or acentrally, stiffness of the hypothesized channel-cytoskeleton tether, and bending modulus of the membrane. Membrane forces are perpendicular to the directions of the principal curvatures of the deformed membrane. Our approach allows for a fine vectorial picture of the local forces gating the channel; membrane forces change with the membrane curvature and are themselves sufficient to affect the open probability of the channel.

Accurate label-free protein quantitation with high- and low-resolution mass spectrometers.

Label-free quantitation of proteins analyzed by tandem mass spectrometry uses either integrated peak intensity from the parent-ion mass analysis (MS1) or features from fragment-ion analysis (MS2), such as spectral counts or summed fragment-ion intensity. We directly compared MS1 and MS2 quantitation by analyzing human protein standards diluted into Escherichia coli extracts on an Orbitrap mass spectrometer. We found that summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity. We compared these results to those obtained from two low-resolution ion-trap mass spectrometers; summed MS2 intensities from LTQ and LTQ Velos instruments were similar in accuracy to those from the Orbitrap. Data from all three instruments are available via ProteomeXchange with identifier PXD000602. Abundance measurements using MS1 or MS2 intensities had limitations, however. While measured protein concentration was on average well-correlated with the known concentration, there was considerable protein-to-protein variation. Moreover, not all human proteins diluted to a mole fraction of 10(-3) or lower were detected, with a strong falloff below 10(-4) mole fraction. These results show that MS1 and MS2 intensities are simple measures of protein abundance that are on average accurate but should be limited to quantitation of proteins of intermediate to higher fractional abundance.

The Geminin and Idas coiled coils preferentially form a heterodimer that inhibits Geminin function in DNA replication licensing.

Geminin is an important regulator of proliferation and differentiation in metazoans, which predominantly inhibits the DNA replication licensing factor Cdt1, preventing genome over-replication. We show that Geminin preferentially forms stable coiled-coil heterodimers with its homologue, Idas. In contrast to Idas-Geminin heterodimers, Idas homodimers are thermodynamically unstable and are unlikely to exist as a stable macromolecule under physiological conditions. The crystal structure of the homology regions of Idas in complex with Geminin showed a tight head-to-head heterodimeric coiled-coil. This Idas-Geminin heterodimer binds Cdt1 less strongly than Geminin-Geminin, still with high affinity (∼30 nm), but with notably different thermodynamic properties. Consistently, in Xenopus egg extracts, Idas-Geminin is less active in licensing inhibition compared with a Geminin-Geminin homodimer. In human cultured cells, ectopic expression of Idas leads to limited over-replication, which is counteracted by Geminin co-expression. The properties of the Idas-Geminin complex suggest it as the functional form of Idas and provide a possible mechanism to modulate Geminin activity.