Microtubule-binding Domain Research Articles

Drug Discovery and Screening Tool Development for Tauopathies by Focusing on Pathogenic Tau Repeat 3 Oligomers.

Comprehending early amyloidogenesis is essential for the development of effective therapeutic strategies. In tauopathies like Alzheimer's disease (AD), the abnormal accumulation of tau protein is initiated by pathological tau seeds. Mounting evidence implies that the microtubule binding domain, consisting of three to four repeats, plays a pivotal role in this process, yet the exact region driving the formation of pathogenic species needs to be further scrutinized. Here, we chemically synthesized individual tau repeats to identify those exhibiting pathogenic prion-like characteristics. Notably, repeat 3 (R3) displayed a remarkable propensity to polymerize, form toxic filaments, and induce cognitive impairment, even in the absence of an aggregation-promoting inducer, highlighting its physiological relevance. Additionally, oligomeric R3 was identified as a particularly pathological form, prompting the establishment of a screening platform. Through screening, tolcapone was found to possess therapeutic efficacy against pathological tau aggregates in PS19 transgenic mice. This screening platform provides a valuable avenue for identifying compounds that selectively interact with peptides implicated in the progression of tauopathies.

Structure of the microtubule anchoring factor NEDD1 bound to the γ-tubulin ring complex.

The γ-tubulin ring complex (γ-TuRC) is an essential multiprotein assembly, in which γ-tubulin, GCP2-6, actin, MZT1 and MZT2 form an asymmetric cone-shaped structure that provides a template for microtubule nucleation. The γ-TuRC is recruited to microtubule organizing centers (MTOCs), such as centrosomes and pre-existing mitotic spindle microtubules, via the evolutionarily-conserved attachment factor NEDD1. NEDD1 contains an N-terminal WD40 domain that binds to microtubules, and a C-terminal domain that associates with the γ-TuRC. However, the structural basis of the NEDD1-γ-TuRC interaction is not known. Here, we report cryo-electron microscopy (cryo-EM) structures of NEDD1 bound to the human γ-TuRC in the absence or presence of the activating factor CDK5RAP2, which interacts with GCP2 to induce conformational changes in the γ-TuRC and promote its microtubule nucleating function. We found that the C-terminus of NEDD1 forms a tetrameric α-helical assembly that contacts the lumen of the γ-TuRC cone, is anchored to GCP4, 5 and 6 via protein modules consisting of MZT1 & GCP3 subcomplexes, and orients its microtubule-binding WD40 domains away from the complex. We biochemically tested our structural models by identifying NEDD1 mutants unable to pull-down γ -tubulin from cultured cells. The structure of the γ-TuRC simultaneously bound to NEDD1 and CDK5RAP2 reveals that both factors can associate with the "open" conformation of the complex. Our results show that NEDD1 does not induce conformational changes in the γ-TuRC, but suggest that anchoring of γ-TuRC-capped microtubules by NEDD1 would be structurally compatible with the significant conformational changes experienced by the γ-TuRC during microtubule nucleation.

CAMSAP3 forms dimers via its α-helix domain that directly stabilize non-centrosomal microtubule minus ends.

Microtubules are vital components of the cytoskeleton. Their plus ends are dynamic and respond to changes in cell morphology, while the minus ends are stable and serve a crucial role in microtubule seeding and maintaining spatial organization. In mammalian cells, the calmodulin-regulated spectrin-associated proteins (CAMSAPs), play a key role in directly regulating the dynamics of non-centrosomal microtubules minus ends. However, the molecular mechanisms are not yet fully understood. Our study reveals that CAMSAP3 forms dimers through its C-terminal α-helix; this dimerization not only enhances the microtubule-binding affinity of the CKK domain but also enables the CKK domain to regulate the dynamics of microtubules. Furthermore, CAMSAP3 also specializes in decorating at the minus end of microtubules through the combined action of the microtubule-binding domain (MBD) and the C-terminal α-helix, thereby achieving dynamic regulation of the minus ends of microtubules. These findings are crucial for advancing our understanding and treatment of diseases associated with non-centrosomal microtubules.

Pathogenic Mutation ΔK280 Promotes Hydrophobic Interactions Involving Microtubule-Binding Domain and Enhances Liquid-Liquid Phase Separation of Tau.

Liquid-liquid phase separation (LLPS) of tau protein can initiate its aggregation which is associated with Alzheimer's disease. The pathogenic mutation ΔK280 can enhance the aggregation of K18, a truncated tau variant comprising the microtubule-binding domain. However, the impact of ΔK280 on K18 LLPS and underlying mechanisms are largely unexplored. Herein, the conformational ensemble and LLPS of ΔK280 K18 through multiscale molecular simulations and microscopy experiments are investigated. All-atom molecular dynamic simulations reveal that ΔK280 significantly enhances the collapse degree and β-sheet content of the K18 monomer, indicating that ΔK280 mutation may promote K18 LLPS, validated by coarse-grained phase-coexistence simulations and microscopy experiments. Importantly, ΔK280 mutation promotes β-sheet formation of six motifs (especially PHF6), increases the hydrophobic solvent exposure of PHF6* and PHF6, and enhances hydrophobic, hydrogen bonding, and cation-π interactions involving most of the motifs, thus facilitating the phase separation of K18. Notably, ΔK280 alters the interaction network among the six motifs, inducing the formation of K18 conformations with high β-sheet contents and collapse degree. Coarse-grained simulations on full-length tau reveal that ΔK280 promotes tau LLPS by enhancing the hydrophobic interactions involving the microtubule-binding domain. These findings offer detailed mechanistic insights into ΔK280-induced tau pathogenesis, providing potential targets for therapeutic intervention.

Structural analysis of microtubule binding by minus-end targeting protein Spiral2

Microtubules (MTs) are dynamic cytoskeletal polymers that play a critical role in determining cell polarity and shape. In plant cells, acentrosomal MTs are localized on the cell surface and are referred to as cortical MTs. Cortical MTs nucleate in the cell cortex and detach from nucleation sites. The released MT filaments perform treadmilling, with the plus-ends of MTs polymerizing and the minus-ends depolymerizing. Minus-end targeting proteins, -TIPs, include Spiral2, which regulates the minus-end dynamics of acentrosomal MTs. Spiral2 accumulates autonomously at MT minus-ends and inhibits filament shrinkage, but the mechanism by which Spiral2 specifically recognizes minus-ends of MTs remains unknown. Here we describe the crystal structure of Spiral2's N-terminal MT-binding domain. The structural properties of this domain resemble those of the HEAT repeat structure of the tumor overexpressed gene (TOG) domain, but the number of HEAT repeats is different and the conformation is highly arched. Gel filtration and co-sedimentation analyses demonstrate that the domain binds preferentially to MT filaments rather than the tubulin dimer, and that the tubulin-binding mode of Spiral2 via the basic surface is similar to that of the TOG domain. We constructed an in silico model of the Spiral2-tubulin complex to identify residues that potentially recognize tubulin. Mutational analysis revealed that the key residues inferred in the model are involved in microtubule recognition, and provide insight into the mechanism by which end-targeting proteins stabilize MT ends.

MAPT haplotype-associated transcriptomic changes in progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is a neurodegenerative movement and cognitive disorder characterized by abnormal accumulation of the microtubule-associated protein tau in the brain. Biochemically, inclusions in PSP are enriched for tau proteoforms with four microtubule-binding domain repeats (4R), an isoform that arises from alternative tau pre-mRNA splicing. While preferential aggregation and reduced degradation of 4R tau protein is thought to play a role in inclusion formation and toxicity, an alternative hypothesis is that altered expression of tau mRNA isoforms plays a causal role. This stems from the observation that PSP is associated with common variation in the tau gene (MAPT) at the 17q21.31 locus which contains low copy number repeats flanking a large recurrent genomic inversion. The complex genomic structural changes at the locus give rise to two dominant haplotypes, termed H1 and H2, that have the potential to markedly influence gene expression. Here, we explored haplotype-dependent differences in gene expression using a bulk RNA-seq dataset derived from human post-mortem brain tissue from PSP (n = 84) and controls (n = 77) using a rigorous computational pipeline, including alternative pre-mRNA splicing. We found 3579 differentially expressed genes in the temporal cortex and 10,011 in the cerebellum. We also found 7214 differential splicing events in the temporal cortex and 18,802 in the cerebellum. In the cerebellum, total tau mRNA levels and the proportion of transcripts encoding 4R tau were significantly increased in PSP compared to controls. In the temporal cortex, the proportion of reads that expressed 4R tau was increased in cases compared to controls. 4R tau mRNA levels were significantly associated with the H1 haplotype in the temporal cortex. Further, we observed a marked haplotype-dependent difference in KANSL1 expression that was strongly associated with H1 in both brain regions. These findings support the hypothesis that sporadic PSP is associated with haplotype-dependent increases in 4R tau mRNA that might play a causal role in this disorder.

Registry alteration in Dynein's microtubule-binding domain: A AAA domain-guided event

Dynein, a motor protein, harnesses chemical energy from ATP hydrolysis to generate mechanical output as it travels along microtubular tracks. Essential to this process is the microtubule-binding domain (MTBD), which facilitates the interaction with and detachment from microtubules. Previous studies have proposed that the mechanism governing this interaction is primarily driven by the coiled-coil stalk attached to the MTBD. However, conflicting arguments suggest the presence of two-way communications, where the binding and unbinding mechanisms may also influence the nucleotide state of dynein. In this study, we employed all-atom explicit solvent simulations, enhanced sampling techniques, and coarse-grained methodologies to systematically investigate the effects of stalk and MT on the structural stabilities of different conformations of MTBD and their sequence of events during the transition from one to another. We found that the globular MTBD domain without stalk and MT predominantly resides in its weak binding configuration. Upon introduction of a limited length of stalk and interaction with MT, a balance between the strong and weak binding configurations is restored. Further introduction of the full-length stalk and interaction with MT strengthen the kinetic stability of these two configurations. We have also explored the sequence of events of the relevant transition between these two states using coarse-grained simulation protocols both in the presence and absence of interaction with MT. In the absence of MT interactions, we found a mixed conformational state of the system that corresponds to the already published crystal structure of Dynein. In the presence of MT, the conformational change of the stalk precedes the conformational change of the MTBD globular domain. Our findings support the view that the nucleotide state of the AAA+ ring determines the state of the MTBD domain through stalk, shedding light on the intricate mechanisms governing dynein's interaction with microtubules.

Synaptic gene expression changes in frontotemporal dementia due to the MAPT 10+ 16 mutation.

Mutations in the MAPT gene encoding tau protein can cause autosomal dominant neurodegenerative tauopathies including frontotemporal dementia (often with Parkinsonism). In Alzheimer's disease, the most common tauopathy, synapse loss is the strongest pathological correlate of cognitive decline. Recently, Positron Emission Tomography (PET) imaging with synaptic tracers revealed clinically relevant loss of synapses in primary tauopathies; however, the molecular mechanisms leading to synapse degeneration in primary tauopathies remain largely unknown. In this study, we examined post-mortem brain tissue from people who died with frontotemporal dementia with tau pathology (FTDtau) caused by the MAPT intronic exon 10+ 16 mutation, which increases splice variants containing exon 10 resulting in higher levels of tau with four microtubule-binding domains. We used RNA sequencing and histopathology to examine temporal cortex and visual cortex, to look for molecular phenotypes compared to age, sex and RNA integrity matched participants who died without neurological disease (n= 12 FTDtau10+ 16 and 13 controls). Bulk tissue RNA sequencing reveals substantial downregulation of gene expression associated with synaptic function. Upregulated biological pathways in human MAPT 10+ 16 brain included those involved in transcriptional regulation, DNA damage response and neuroinflammation. Histopathology confirmed increased pathological tau accumulation in FTDtau10 + 16 cortex as well as a loss of presynaptic protein staining and region-specific increased colocalization of phospho-tau with synapses in temporal cortex. Our data indicate that synaptic pathology likely contributes to pathogenesis in FTDtau10 + 16 caused by the MAPT 10+ 16 mutation.

Stepping dynamics of dynein characterized by MINFLUX.

Cytoplasmic dynein is a dimeric motor that drives minus-end directed transport on microtubules (MTs). To couple ATP hydrolysis to a mechanical step, a dynein monomer must be released from the MT before undergoing a conformational change that generates a bias towards the minus end. However, the dynamics of dynein stepping have been poorly characterized by tracking flexible regions of the motor with limited resolution. Here, we developed a cysteine-light mutant of yeast dynein and site-specifically labeled its MT-binding domain in vitro. MINFLUX tracking at sub-millisecond resolution revealed that dynein hydrolyzes one ATP per step and takes multitudes of 8 nm steps at physiological ATP. Steps are preceded by the transient movement towards the plus end. We propose that these backward "dips" correspond to MT release and subsequent diffusion of the stepping monomer around its MT-bound partner before taking a minus-end-directed conformational change of its linker. Our results reveal the order of sub-millisecond events that result in a productive step of dynein.

Regulatable assembly of synthetic microtubule architectures using engineered microtubule-associated protein-IDR condensates

Microtubule filaments are assembled into higher-order structures using microtubule-associated proteins. However, synthetic MAPs that direct the formation of new structures are challenging to design, as nanoscale biochemical activities must be organized across micron length-scales. Here, we develop modular MAP-IDR condensates (synMAPs) that enable inducible assembly of higher-order microtubule structures for synthetic exploration invitro and in mammalian cells. synMAPs harness a small microtubule-binding domain from oligodendrocytes (TPPP) whose activity we show can be rewired by interaction with unrelated condensate-forming IDR sequences. This combination is sufficient to allow synMAPs to self-organize multivalent structures that bind and bridge microtubules into higher-order architectures. By regulating the connection between the microtubule-binding domain and condensate-forming components of a synMAP, the formation of these structures can be triggered by small molecules or cell-signaling inputs. We systematically test a panel of synMAP circuit designs to define how the assembly of these synthetic microtubule structures can be controlled at the nanoscale (via microtubule-binding affinity) and microscale (via condensate formation). synMAPs thus provide a modular starting point for the design of higher-order microtubule systems and an experimental testbed for exploring condensate-directed mechanisms of higher-order microtubule assembly from the bottom-up.

A force-sensitive mutation reveals a non-canonical role for dynein in anaphase progression.

The diverse roles of the dynein motor in shaping microtubule networks and cargo transport complicate in vivo analysis of its functions significantly. To address this issue, we have generated a series of missense mutations in Drosophila Dynein heavy chain. We show that mutations associated with human neurological disease cause a range of defects, including impaired cargo trafficking in neurons. We also describe a novel microtubule-binding domain mutation that specifically blocks the metaphase-anaphase transition during mitosis in the embryo. This effect is independent from dynein's canonical role in silencing the spindle assembly checkpoint. Optical trapping of purified dynein complexes reveals that this mutation only compromises motor performance under load, a finding rationalized by the results of all-atom molecular dynamics simulations. We propose that dynein has a novel function in anaphase progression that depends on it operating in a specific load regime. More broadly, our work illustrates how in vivo functions of motors can be dissected by manipulating their mechanical properties.

Depletion of TDP-43 exacerbates tauopathy-dependent brain atrophy by sensitizing vulnerable neurons to caspase 3-mediated endoproteolysis of tau in a mouse model of Multiple Etiology Dementia.

TDP-43 proteinopathy, initially disclosed in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), coexists with tauopathy in a variety of neurodegenerative disorders, termed multiple etiology dementias (MEDs), including Alzheimer's Disease (AD). While such co-pathology of TDP-43 is strongly associated with worsened neurodegeneration and steeper cognitive decline, the pathogenic mechanism underlying the exacerbated neuron loss remains elusive. The loss of TDP-43 splicing repression that occurs in presymptomatic ALS-FTD individuals suggests that such early loss could facilitate the pathological conversion of tau to accelerate neuron loss. Here, we report that the loss of TDP-43 repression of cryptic exons in forebrain neurons (CaMKII-CreER;Tardbp f/f mice) is necessary to exacerbate tauopathy-dependent brain atrophy by sensitizing vulnerable neurons to caspase 3-dependent cleavage of endogenous tau to promote tauopathy. Corroborating this finding within the human context, we demonstrate that loss of TDP-43 function in iPSC-derived cortical neurons promotes early cryptic exon inclusion and subsequent caspase 3-mediated endoproteolysis of tau. Using a genetic approach to seed tauopathy in CaMKII-CreER;Tardbp f/f mice by expressing a four-repeat microtubule binding domain of human tau, we show that the amount of tau seed positively correlates with levels of caspase 3-cleaved tau. Importantly, we found that the vulnerability of hippocampal neurons to TDP-43 depletion is dependent on the amount of caspase 3-cleaved tau: from most vulnerable neurons in the CA2/3, followed by those in the dentate gyrus, to the least in CA1. Taken together, our findings strongly support the view that TDP-43 loss-of-function exacerbates tauopathy-dependent brain atrophy by increasing the sensitivity of vulnerable neurons to caspase 3-mediated endoproteolysis of tau, resulting in a greater degree of neurodegeneration in human disorders with co-pathologies of tau and TDP-43. Our work thus discloses novel mechanistic insights and therapeutic targets for human tauopathies harboring co-pathology of TDP-43 and provides a new MED model for testing therapeutic strategies.

Pick's Disease Presenting as Tremulous Parkinsonism with Limited Levodopa Response-A Rare Cause of Corticobasal Syndrome.

Corticobasal syndrome is a clinical diagnosis and common pathological causes are corticobasal degeneration, progressive supranuclear palsy and Alzheimer's disease. We would like to highlight a rare but important differential of corticobasal syndrome. A 78-year-old female had a 4-year history of predominantly right-hand rest tremor, worsening of handwriting but no change in cognition. The clinical examination showed right upper limb postural and kinetic tremor, mild wrist rigidity and reduced amplitude of right-sided finger tapping. She was initially diagnosed as idiopathic Parkinson's disease. Five years after onset of symptoms, she demonstrated bilateral myoclonic jerks and right upper limb dystonic posturing. She could not copy movements with the right hand. The magnetic resonance imaging (MRI) revealed disproportionate atrophy in the parietal lobes bilaterally. The clinical diagnosis was changed to probable corticobasal syndrome. She passed away 11 years from onset of symptoms at the age of 85 years. She underwent a post-mortem. The anterior and posterior frontal cortex, anterior cingulate, temporal neocortex, hippocampus and amygdaloid complex demonstrated considerable tau-related pathology consisting of a dense background of neuropil threads, and rounded, paranuclear neuronal inclusions consistent with Pick bodies. The immunostaining for three microtubule binding domain repeats (3R) tau performed on sections from the frontal and temporal lobes, basal ganglia and midbrain highlighted several inclusions whilst no 4R tau was observed. She was finally diagnosed with Pick's disease. Pick's disease can rarely present with clinical features of corticobasal syndrome.

Crystal structure of human Cep57 C-terminal domain reveals the presence of leucine zipper and the potential microtubule binding region.

Cep57, a vital centrosome-associated protein, recruits essential regulatory enzymes for centriole duplication. Its dysfunction leads to anomalies, including reduced centrioles and mosaic-variegated aneuploidy syndrome. Despite functional investigations, understanding structural aspects and their correlation with functions is partial till date. We present the structure of human Cep57 C-terminal microtubule binding (MT-BD) domain, revealing conserved motifs ensuring functional preservation across evolution. A leucine zipper, with an adjacent possible microtubule-binding region, potentially forms a stabilizing scaffold for microtubule nucleation-accommodating pulling and tension from growing microtubules. This study highlights conserved structural features of Cep57 protein, compares them with other analogous proteins, and explores how protein function is maintained across diverse organisms.

The Shot CH1 domain recognises a distinct form of F-actin during Drosophila oocyte determination.

In Drosophila, only one cell in a multicellular female germline cyst is specified as an oocyte and a similar process occurs in mammals. The symmetry-breaking cue for oocyte selection is provided by the fusome, a tubular structure connecting all cells in the cyst. The Drosophila spectraplakin Shot localises to the fusome and translates its asymmetry into a polarised microtubule network that is essential for oocyte specification, but how Shot recognises the fusome is unclear. Here, we demonstrate that the actin-binding domain (ABD) of Shot is necessary and sufficient to localise Shot to the fusome and mediates Shot function in oocyte specification together with the microtubule-binding domains. The calponin homology domain 1 of the Shot ABD recognises fusomal F-actin and requires calponin homology domain 2 to distinguish it from other forms of F-actin in the cyst. By contrast, the ABDs of utrophin, Fimbrin, Filamin, Lifeact and F-tractin do not recognise fusomal F-actin. We therefore propose that Shot propagates fusome asymmetry by recognising a specific conformational state of F-actin on the fusome.

Abstract 2009: Novel clinically relevant CLIP-170 variants display an extended conformation microtubule-lattice bound conformation associated with taxane resistance

Abstract Taxanes are microtubule (MT)-stabilizing drugs widely used for the treatment of solid tumors, including the highly prevalent cancers of the breast, the prostate and gastric cancer. However, despite their initial activity, taxane resistance invariably occurs, leaving patients with limited therapeutic options. Currently, the molecular determinants of clinical taxane resistance remain poorly understood. We recently identified CLIP-170S, a novel truncated variant of the MT plus-end binding protein CLIP-170, that confers taxane resistance in vitro and is associated with clinical taxane resistance in patients with gastric cancer. We further showed that CLIP-170S is aberrantly localized along the MT lattice, thus impairing taxane access to its binding site in the MT lumen. However, the mechanism underlying the resistance-inducing extensive lattice localization of CLIP-170S is unknown. The activity and MT binding properties of canonical CLIP-170 are regulated by conformational changes mediated by the interaction of its N- and C-terminal domains. In its inactive, “closed” conformation, CLIP-170 is soluble as the N- and C-terminal domains interact with each other. In the active “open” conformation, CLIP-170’s N- and C-terminal domains are apart, thus exposing its MT-binding domain and allowing MT plus-end binding. As CLIP-170S is missing part of its N-terminal domain, we hypothesized that CLIP-170S is constitutively displayed in an open conformation that promotes its extensive and aberrant localization to the MT lattice. To prove this hypothesis, we used the FKBP-rapalog-FRB system to “force” CLIP-170S to assume a “closed” conformation. We generated a fusion CLIP-170S protein tagged with FKBP and FRB domains in its N- and C-terminal, respectively. The addition of rapalog induced FKBP (N-ter) and FRB (C-ter) domains to interact, promoting CLIP-170S to adopt a “closed” conformation. CLIP-170S in this “closed” conformation resulted in its release from the MT-lattice and restored taxane binding to MTs. We next extended our search to other CLIP-170 clinically prevalent variants with sequence alteration predicted to bind to the MT-lattice and potentially interfere with taxane sensitivity. This search identified CLIP-170IR (an intron retention variant) and several other truncation mutants prevalent in gastric, breast and lung cancer cell lines. Experimentally, we showed that these variants not only phenocopy CLIP-170S binding to the MT- lattice but also confer taxane-resistance. Together, these data demonstrate a novel mechanism of taxane resistance mediated by CLIP-170 mutations that induce CLIP-170 to adopt a constitutively “open” MT-lattice binding conformation, which renders them susceptible to causing taxane resistance. Citation Format: Urko del Castillo, Kiran Kumari Sahu, William G. Stone, Manish A. Shah, Paraskevi Giannakakou. Novel clinically relevant CLIP-170 variants display an extended conformation microtubule-lattice bound conformation associated with taxane resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2009.

Abstract 1647: Plk4 phosphorylates the distal tip complex to promote centriole growth

Abstract The centrosome is the major microtubule-organizing center of the cell with a key role in forming the mitotic spindle. The centrosome consists of two core duplicating elements, the centrioles, surrounded by pericentriolar material, a cloud of proteins that nucleates microtubules. Centrioles are barrel-like structures composed of microtubule bundles and are essential to maintain centrosome number. Therefore, centriole duplication and growth are tightly regulated to ensure proper centrosome number throughout the cell cycle. Alterations in centrosome number lead to an abnormally shaped mitotic spindle that often mis-segregate chromosomes, thereby causing chromosomal instability, a hallmark of cancer. Defects in centriole growth, such as over-elongation, can increase centrosome numbers (known as ‘centriole amplification’, an overproduction of centrioles frequently observed in cancer cells). For example, over-elongation of the centriolar barrel can cause multiple ectopic daughter centrioles to assemble or simply fragment, producing multiple smaller (but functional) centrioles. Thus, understanding how centrioles grow and define length is a major question in the field. Centrioles elongate at their distal ends and, in Drosophila, elongation is regulated by a group of proteins, Cep97, CP110 and Klp10A. Elongation is negatively regulated by Cep97 and CP110 which cap centrioles at their distal end to maintain centriole length, by counteracting the microtubule depolymerizing kinesin, Klp10A. Recently, we identified a new centriole regulator, Cep104, that also localizes to the centriole distal tip. We discovered that Cep104 promotes centriole elongation through its microtubule-binding TOG domain. Additionally, Cep104 localization to the centriole distal ends is dependent on Cep97. Furthermore, Cep97, CP110, Cep104 and Klp10A form a complex and we have mapped their sites of interaction. Collectively, we refer to this as the Distal Tip Complex (DTC). Although the DTC is first recruited to new daughter centrioles during G1/S-phase, the majority of centriole elongation occurs during mitosis. Here, we explore the mechanisms that regulate the DTC to promote centriole growth during mitosis. We hypothesize that Polo-like kinase 4 (Plk4), the master regulator of centriole duplication, phosphorylates and inhibits the DTC to promote elongation of nascent centrioles. Notably, we found that centrioles are longer in cells overexpressing Plk4. Using tandem mass spectrometry, we identified multiple sites that Plk4 phosphorylates on each DTC protein. Analysis of DTC phosphomutants revealed that the phosphorylation state of Klp10A, Cep97 and CP110 affect centriole number. We are currently investigating how Plk4 phosphorylation regulates the activities of these conserved centriole length factors. Citation Format: Anastasia Amoiroglou, John M. Ryniawec, Daniel W. Buster, Gregory C. Rogers. Plk4 phosphorylates the distal tip complex to promote centriole growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1647.

Kinetic Ensemble of Tau Protein through the Markov State Model and Deep Learning Analysis.

The ordered assembly of Tau protein into filaments characterizes Alzheimer's and other neurodegenerative diseases, and thus, stabilization of Tau protein is a promising avenue for tauopathies therapy. To dissect the underlying aggregation mechanisms on Tau, we employ a set of molecular simulations and the Markov state model to determine the kinetics of ensemble of K18. K18 is the microtubule-binding domain of Tau protein and plays a vital role in the microtubule assembly, recycling processes, and amyloid fibril formation. Here, we efficiently explore the conformation of K18 with about 150 μs lifetimes in silico. Our results observe that all four repeat regions (R1-R4) are very dynamic, featuring frequent conformational conversion and lacking stable conformations, and the R2 region is more flexible than the R1, R3, and R4 regions. Additionally, it is worth noting that residues 300-310 in R2-R3 and residues 319-336 in R3 tend to form sheet structures, indicating that K18 has a broader functional role than individual repeat monomers. Finally, the simulations combined with Markov state models and deep learning reveal 5 key conformational states along the transition pathway and provide the information on the microsecond time scale interstate transition rates. Overall, this study offers significant insights into the molecular mechanism of Tau pathological aggregation and develops novel strategies for both securing tauopathies and advancing drug discovery.

Molecular Insights into the Differential Effects of Acetylation on the Aggregation of Tau Microtubule-Binding Repeats.

Aggregation of tau protein into intracellular fibrillary inclusions is characterized as the hallmark of tauopathies, including Alzheimer's disease and chronic traumatic encephalopathy. The microtubule-binding (MTB) domain of tau, containing either three or four repeats with sequence similarities, plays an important role in determining tau's aggregation. Previous studies have reported that abnormal acetylation of lysine residues displays a distinct effect on the formation of pathological tau aggregates. However, the underlying molecular mechanism remains mostly elusive. In this study, we performed extensive replica exchange molecular dynamics (REMD) simulations of 144 μs in total to systematically investigate the dimerization of four tau MTB repeats and explore the impacts of Lys280 (K280) or Lys321 (K321) acetylation on the conformational ensembles of the R2 or R3 dimer. Our results show that R3 is the most prone to aggregation among the four repeats, followed by R2 and R4, while R1 displays the weakest aggregation propensity with a disordered structure. Acetylation of K280 could promote the aggregation of R2 peptides by increasing the formation of β-sheet structures and strengthening the interchain interaction. However, K321 acetylation decreases the β-sheet content of the R3 dimer, reduces the ability of R3 peptides to form long β-strands, and promotes the stable helix structure formation. The salt bridge and Y310-Y310 π-π stacking interactions of the R3 dimer are greatly weakened by K321 acetylation, resulting in the inhibition of dimerization. This study uncovers the structural ensembles of tau MTB repeats and provides mechanistic insights into the influences of acetylation on tau aggregation, which may deepen the understanding of the pathogenesis of tauopathies.

A structural and dynamic visualization of the interaction between MAP7 and microtubules

Microtubules (MTs) are key components of the eukaryotic cytoskeleton and are essential for intracellular organization, organelle trafficking and mitosis. MT tasks depend on binding and interactions with MT-associated proteins (MAPs). MT-associated protein 7 (MAP7) has the unusual ability of both MT binding and activating kinesin-1-mediated cargo transport along MTs. Additionally, the protein is reported to stabilize MTs with its 112 amino-acid long MT-binding domain (MTBD). Here we investigate the structural basis of the interaction of MAP7 MTBD with the MT lattice. Using a combination of solid and solution-state nuclear magnetic resonance (NMR) spectroscopy with electron microscopy, fluorescence anisotropy and isothermal titration calorimetry, we shed light on the binding mode of MAP7 to MTs at an atomic level. Our results show that a combination of interactions between MAP7 and MT lattice extending beyond a single tubulin dimer and including tubulin C-terminal tails contribute to formation of the MAP7-MT complex.