Cep164 triggers ciliogenesis by recruiting Tau tubulin kinase 2 to the mother centriole

Primary cilia play critical roles in development and disease. Their assembly is triggered by mature centrioles (basal bodies) and requires centrosomal protein 164kDa (Cep164), a component of distal appendages. Here we show that loss of Cep164 leads to early defects in ciliogenesis, reminiscent of the phenotypic consequences of mutations in TTBK2 (Tau tubulin kinase 2). We identify Cep164 as a likely physiological substrate of TTBK2 and demonstrate that Cep164 and TTBK2 form a complex. We map the interaction domains and demonstrate that complex formation is crucial for the recruitment of TTBK2 to basal bodies. Remarkably, ciliogenesis can be restored in Cep164-depleted cells by expression of chimeric proteins in which TTBK2 is fused to the C-terminal centriole-targeting domain of Cep164. These findings indicate that one of the major functions of Cep164 in ciliogenesis is to recruit active TTBK2 to centrioles. Once positioned, TTBK2 then triggers key events required for ciliogenesis, including removal of CP110 and recruitment of intraflagellar transport proteins. In addition, our data suggest that TTBK2 also acts upstream of Cep164, contributing to the assembly of distal appendages.The primary cilium (PC) functions as an antenna-like signaling organelle typically found on postmitotic cells (1–3). It consists of a microtubule-based axoneme enclosed within a ciliary membrane, and its assembly is triggered at the basal body (4, 5). The basal body, in turn, is derived from one of the two centrioles that make up the centrosome, specifically the mature (or “mother”) centriole (M centriole) (6–9). Long erroneously considered a vestigial organelle, the PC has emerged as a key structure for sensing extracellular stimuli and hence plays crucial roles in cellular responses to both mechanical and chemical cues. In vertebrates, PC function has been linked to the regulation of many important aspects of embryonic development as well as tissue homeostasis in adulthood (2, 10); moreover, defects in ciliary assembly or function have been linked to a large number of human diseases known as ciliopathies (3, 6, 11–13).PC structure and formation have long been studied at a morphological level (14, 15), but a molecular understanding of the regulation of PC assembly and resorption is only beginning to emerge (5, 7, 8, 16, 17). In cultured cells, PC formation generally occurs when cells exit the cell cycle to enter quiescence (Go), and, conversely, PCs are often resorbed when cells reenter the cell cycle. Thus, PC formation can readily be triggered by serum starvation of some cultured cells, including telomerase-immortalized retinal pigment epithelial (RPE-1) cells. Early steps in PC formation include the docking of membrane vesicles to centrioles (14, 18, 19), the removal of the capping protein CP110 from the distal part of the M centriole (20, 21), the recruitment of intraflagellar transport (IFT) protein complexes (22–24), the formation of a transition zone at the membrane (25, 26), and, finally, the outgrowth of the ciliary axoneme (27, 28).An important role in PC formation resides with specific components of the M centriole, termed distal appendages (14, 15). These appendages are considered critical for the early docking of Golgi-derived membrane vesicles and the subsequent anchorage of the basal body underneath the plasma membrane. Following the discovery of centrosomal protein 164kDa (Cep164), the first marker for distal appendages (29), several additional distal appendage proteins (DAPs) have recently been identified and functionally linked to ciliogenesis. These include centrosomal protein 83kDa (Cep83)/CCDC41, centrosomal protein 89kDa (Cep89)/CCDC123, SCLT1, and FBF1 (18, 19, 30, 31). The DAP Cep164 was discovered in a screen for components that are critical for PC formation (29). Subsequently, mutations in Cep164 were linked to ciliopathies, providing direct proof for the importance of this protein in human pathophysiology (32). At a mechanistic level, Cep164 was shown to be required at an early stage of PC formation, notably for the docking of membrane vesicles to the basal body (18). Moreover, two components of the vesicle transport machinery, the small GTPase Rab8 and its guanine–nucleotide exchange factor Rabin8, were identified as interaction partners of Cep164 (18). Despite this progress, the precise molecular functions of Cep164 remain to be fully understood.Importantly, protein kinases have also been implicated in ciliogenesis and in cilia-related diseases. These include Nek1 and Nek8, two members of the family of NIMA-related kinases (33, 34), and Tau Tubulin Kinase 2 (TTBK2), a member of the casein kinase 1 family (35–37).Here, we report that Cep164 and TTBK2 form a complex and that formation of this complex at M centrioles is essential for ciliogenesis. We show that the noncatalytic C-terminal domain of TTBK2 interacts with Cep164 and that formation of the complex critically depends on the WW domain within the N-terminal domain of Cep164. We also provide evidence that Cep164 is a likely physiological substrate of TTBK2. Use of chimeric TTBK2–Cep164 constructs in siRNA-rescue experiments leads us to conclude that a main function of Cep164 consists of the recruitment of TTBK2 to M centrioles. Once localized correctly, TTBK2 is then in a position to trigger PC formation through phosphorylation of appropriate substrates. Interestingly, our data also reveal a role for TTBK2 in the assembly of distal appendages. Overexpression of active kinase in fact enhances not only the association of DAPs with existing appendages but also triggers their occasional recruitment to daughter centrioles (D centrioles).     

C2cd3 is critical for centriolar distal appendage assembly and ciliary vesicle docking in mammals

The primary cilium plays critical roles in vertebrate development and physiology, but the mechanisms underlying its biogenesis remain poorly understood. We investigated the molecular function of C2 calcium-dependent domain containing 3 (C2cd3), an essential regulator of primary cilium biogenesis. We show that C2cd3 is localized to the centriolar satellites in a microtubule- and Pcm1-dependent manner; however, C2cd3 is dispensable for centriolar satellite integrity. C2cd3 is also localized to the distal ends of both mother and daughter centrioles and is required for the recruitment of five centriolar distal appendage proteins: Sclt1, Ccdc41, Cep89, Fbf1, and Cep164. Furthermore, loss of C2cd3 results in failure in the recruitment of Ttbk2 to the ciliary basal body as well as the removal of Cp110 from the ciliary basal body, two critical steps in initiating ciliogenesis. C2cd3 is also required for recruiting the intraflagellar transport proteins Ift88 and Ift52 to the mother centriole. Consistent with a role in distal appendage assembly, C2cd3 is essential for ciliary vesicle docking to the mother centriole. Our results suggest that C2cd3 regulates cilium biogenesis by promoting the assembly of centriolar distal appendages critical for docking ciliary vesicles and recruiting other essential ciliogenic proteins.The primary cilium is a fundamentally important organelle, the loss of which in humans causes a broad spectrum of genetic disorders known as ciliopathies (1). In addition to sensory and motile functions, the primary cilium plays a central role in the signal transduction of the Hh, PDGF, Wnt, Hippo, and calcium signaling pathways (2–4). Recent genomic and proteomic approaches have identified a ciliome consisting of hundreds of proteins (5, 6). However, the molecular functions of most of these proteins remain elusive.The primary cilium originates from the basal body, a centriole-derived structure (1). A centriole comprises a core multiprotein complex surrounded by a cylinder of nine microtubule triplets. In addition, the oldest centriole, the mother centriole, possesses distal and subdistal appendages. Centrioles and surrounding pericentriolar material constitute the centrosomes and spindle poles in dividing cells (7). In quiescent cells, Golgi-derived ciliary vesicles dock at the distal end of the mother centriole (8). The mother centriole, now known as the basal body, migrates to the cell surface with the docked ciliary vesicle. The ciliary vesicle fuses with the plasma membrane, allowing the cilium to protrude from the cell surface.The distal appendages of the mother centriole, also known as transition fibers of the cilium, are protein complexes comprising at least five components (Ccdc41/Cep83, Cep89/Cep123, Sclt1, Fbf1, and Cep164) (9). Distal appendages are critical for the recruitment of Tau tubulin kinase 2 (Ttbk2), which appears to play a critical role in removing centrosomal protein of 110kD (Cp110, also known as Ccp110, Mouse Genome Informatics, www.informatics.jax.org/marker/MGI:2141942), an inhibitor of ciliogenesis, from the distal end of the mother centriole (10). Recent studies have also suggested that components of the distal appendages are essential for docking ciliary vesicles to the mother centriole (9, 11–14).The axoneme, the microtubule skeleton of the cilium, extends from the distal end of the mother centriole and requires intraflagellar transport (IFT), which was known to mediate cargo movement within the cilium (15). Mutations disrupting the functions of the IFT motors (kinesin II and cytoplasmic dynein) or the IFT cargo adaptor complexes (IFT-A and IFT-B) lead to defects in cilia biogenesis. Recent studies have revealed an essential role for distal appendage proteins in recruiting IFT proteins to the mother centriole, suggesting another mechanism by which centriolar distal appendages promote ciliogenesis (9, 12, 13).Centriolar satellites are electron-dense particles around the centrosome and basal body (16). Many centriolar satellite components have been identified, including Pcm1, the BBSome, a multiprotein complex comprising seven Bardet–Biedl syndrome-related proteins, as well as Cep290 and Ofd1 (17–20). Interestingly, the functions of various centriolar satellite proteins diverge. Pcm1 and the BBSome promote ciliary membrane biogenesis and the trafficking of ciliary membrane proteins (18, 21). Ofd1, on the other hand, appears to regulate ciliogenesis by recruiting components of the distal appendages and IFT particles, although a recent study appeared to suggest an additional negative role of Ofd1 in ciliogenesis (22, 23).Through the study of two loss-of-function mouse mutants, we have previously identified a novel C2 domain-containing protein, C2cd3, as an essential regulator of ciliogenesis and mouse embryonic development (24). In the current study, we demonstrate that C2cd3 is localized to centriolar satellites and that its localization is dependent on Pcm1 and dynein-mediated retrograde transport. C2cd3 is also localized to the distal ends of the mother and daughter centrioles and is required for the recruitment of five distal appendage proteins: Ccdc41, Sclt1, Cep89, Fbf1, and Cep164. Moreover, in the absence of C2cd3, Ttbk2 is not recruited to the distal end of the mother centriole, nor is Cp110 removed. In addition, the recruitment of Ift88 and Ift52, two IFT complex-B components, does not occur in C2cd3 mutants. Finally, we observed that the docking of the ciliary vesicles to the mother centriole is dependent on C2cd3. Our results suggest that C2cd3 regulates the initiation of ciliogenesis through centriolar maturation, ciliogenic protein recruitment and ciliary vesicle docking.     

Conserved TCP domain of Sas-4/CPAP is essential for pericentriolar material tethering during centrosome biogenesis

Pericentriolar material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4’s tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of β-sheets fold. This unique feature of the TCP domain suggests that it could provide an “extended surface-like” platform to tether the Sas-4–PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9–10th β-strands (β9–10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 β9–10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the β9–10 surface in mediating protein–protein interactions for efficient Sas-4–PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication.Centrosomes consist of a pair of centrioles surrounded by a protein network of pericentriolar material (PCM), the main sites for microtubule nucleation and anchoring and thus responsible for PCM’s role as the principle microtubule-organizing centers (MTOCs) of cells (1–4). When PCM is not recruited, centrioles are unstable, and thus no functional centrosomes are generated (5, 6). Although initial proteomic studies suggested PCM to be an amorphous cloud composed of more than a 100 different proteins (7), recent superresolution microscopy of fly and human centrosomes have indicated key centrosomal proteins essential for centrosome biogenesis to be organized into distinct spatial compartments before appearing as a PCM cloud surrounding the centriole (6, 8–11).Thus, there could be a protein providing an interface for mediating PCM tethering to a centriole, a suitable candidate of which is the conserved centrosomal protein Sas-4 (CPAP in human), forming a layer closely associated with the centriole wall and yet shown to interact with various PCM components (6, 12). Functional studies in various model organisms suggest that Sas-4 proteins are required for both centriole formation and PCM assembly (6, 12); in the absence of Sas-4, nascent centrioles form but fail to mature into centrosomes (6). Overexpression of Sas-4 in flies produces PCM-like structures (13), whereas reduced amounts of Sas-4 in worms result in centrosomes having proportionally less PCM (12). Thus, although it is clear that Sas-4 is essential for centrosome biogenesis, the mechanisms by which Sas-4 contributes to PCM assembly remains elusive.During the course of these studies, we and others have reported that Sas-4/CPAP, a protein essential for centriole formation was found to interact with several centrosomal and PCM proteins including Cnn, Asl, D-PLP, γ-TuRC, SIL, Cep135, Cep120, and tubulin dimers (5, 6, 14–16). In Drosophila, the N-terminal domain of Sas-4 provides a scaffolding site for cytoplasmic protein complexes (hereafter referred to as Sas-4–PCM scaffold) and tethers the components of Sas-4–PCM scaffold to a centrosome matrix via its C terminus (6).Interestingly, the C-terminal region of Sas-4 proteins contains a conserved TCP10c domain (Pfam: PF07202) (hereafter referred to as TCP for brevity) (Fig. 1A and SI Appendix, Fig. S1). An E1235V missense mutation within this domain in CPAP has been identified in patients with primary microcephaly (MCPH), resulting in a reduced interaction with STIL (Ana2 in Drosophila), a centriole duplication factor also implicated in MCPH (16–18). Accordingly, recent structural studies on CPAP-STIL complex revealed that CPAP-STIL interaction is required during centriole assembly (19, 20). The C-terminal domain of CPAP has also been shown to mediate an interaction with another MCPH protein Cep135 (Bld-10 in Drosophila) and that interaction is required for centriole assembly. Bld-10 is a core centriolar protein and is required to stabilize structural integrity of centrioles (21–23). Taken together, we therefore speculate that the TCP domain could mediate protein–protein interactions and might serve as a tethering site for Sas-4–PCM scaffold–centriole interactions.Open in a separate windowFig. 1.Crystal structure of Drosophila Sas-4–TCP domain. (A) Domain architecture of Drosophila Sas-4 and its human ortholog CPAP. The fragment used for crystallization is indicated by a black underline. (B) Cartoon view of the overall structure of Sas-4–TCP. The invisible part of β16–20 in the crystal structure is shown as dotted lines. (C) Side view of Sas-4–TCP along the longitudinal axis from the N to C termini. Twisting of the TCP β-strands is diagramed below. F_L, surface left to β1; F_R, surface right to β1. (D and E) Cross-strand ladder residues on F_L (D) and F_R (E) are shown in spheres and classified into different types by color (purple, positively charged residues; red, negatively charged residues; orange, polar residues; green, hydrophobic residues).Although it appears that Sas-4 plays pivotal roles in centriole formation, assembling protein complexes in the cytoplasm, and tethering them to a developing centrosome, the mechanisms by which Sas-4 accomplishes its tethering role have remained unclear. In this study, we therefore investigated the structural basis of Sas-4 and show that via its conserved C-terminal TCP domain, it could provide an “extended surface-like” platform by which Sas-4 could mediate the Sas-4–PCM scaffold–centriole interaction during centrosome biogenesis.     

Epidermal development,growth control,and homeostasis in the face of centrosome amplification

As nucleators of the mitotic spindle and primary cilium, centrosomes play crucial roles in equal segregation of DNA content to daughter cells, coordination of growth and differentiation, and transduction of homeostatic cues. Whereas the majority of mammalian cells carry no more than two centrosomes per cell, exceptions to this rule apply in certain specialized tissues and in select disease states, including cancer. Centrosome amplification, or the condition of having more than two centrosomes per cell, has been suggested to contribute to instability of chromosomes, imbalance in asymmetric divisions, and reorganization of tissue architecture; however, the degree to which these conditions are a direct cause of or simply a consequence of human disease is poorly understood. Here we addressed this issue by generating a mouse model inducing centrosome amplification in a naturally proliferative epithelial tissue by elevating Polo-like kinase 4 (Plk4) expression in the skin epidermis. By altering centrosome numbers, we observed multiciliated cells, spindle orientation errors, and chromosome segregation defects within developing epidermis. None of these defects was sufficient to impart a proliferative advantage within the tissue, however. Rather, impaired mitoses led to p53-mediated cell death and contributed to defective growth and stratification. Despite these abnormalities, mice remained viable and healthy, although epidermal cells with centrosome amplification were still appreciable. Moreover, these abnormalities were insufficient to disrupt homeostasis and initiate or enhance tumorigenesis, underscoring the powerful surveillance mechanisms in the skin.Centrosomes play crucial functions within the cell by organizing microtubules and by participating in the assembly of the primary cilium, an antenna-like structure that senses the cellular environment and transmits signaling cues. On a structural basis, each centrosome consists of orthogonally positioned centrioles and its surrounding protein-rich pericentriolar material (PCM). The majority of mammalian cells contain one centrosome throughout interphase (G1), and then replicate during S-phase of the cell cycle in preparation for mitosis (1). Centrosomes generally template their own duplication, and they keep their numbers in check through tight posttranslational regulation of the duplication process itself. Through licensing mechanisms, centrosomal reduplication is prevented, and by virtue of their role in nucleating a bipolar mitotic spindle, centrosomes are faithfully partitioned into each daughter cell at the end of mitosis (2, 3).Mutations and misregulation of centrosomal proteins have been associated with various human disorders, including ciliopathies, obesity, neurologic disorders, and miscarriages (4–6). Numerical aberrations in centrosome numbers have been demonstrated to alter ciliary signaling and have been proposed to be the underlying cause for certain defects in tissue organization (7, 8). In addition, recent genetic studies on the master regulator of centrosome number, polo-like kinase 4 (PLK4), have shown that, whether too few or too many, perturbations in centrosome numbers can directly impede brain development, leading to microcephaly both in mice and in humans (9, 10). PLK4 mutations also have been associated with mitotic-origin aneuploidy in human embryos, suggestive of a possible link among PLK4, aneuploidy, and pregnancy loss (6).Centrosome amplification, or the condition of having more than a cell’s customary pair of such structures, has garnered attention for more than a century (11). Given the enhanced apoptosis resulting from centrosome amplification in the brain, it is intriguing that centrosome amplification was originally noted for its presence in cancer cells (9). Indeed, increased centrosome number is a hallmark of many cancers, and it correlates with poor clinical prognoses in some malignancies, including those of epithelial origin (12, 13). In flies, centrosomal alterations have been found to expand the pool of proliferative progenitors in serial neuroblast transplantation assays, a phenomenon attributed to an imbalance in asymmetric divisions (14, 15). In mammals, however, despite the strong correlation with hyperproliferative disorders, whether centrosomal abnormalities are the cause, the consequence, or a neutral bystander of cancer remains unclear (16, 17).Based on the assumption that centrosome amplification results in multipolar mitoses, initial research efforts focused on drawing mechanistic links to chromosomal instability (18–20). Surprisingly, however, at least in various cancer cell lines examined in vitro, multipolar divisions turned out to be rare and typically inviable (21). Instead, these cells seemed to have developed strategies to cope with extra centrosomes, including clustering them together such that a bipolar spindle could still form (22–25). That said, even within a bipolar spindle network, chromosome segregation errors involving merotelic attachment have been observed, and these can contribute to chromosomal instability if the mitotic checkpoint is bypassed (21, 26). Taken together, these studies underscore the importance of delving more deeply into the physiological relevance of centrosome amplification in additional mammalian tissues in vivo, and to parse out the centrosomal contribution to tissue function.Mammalian epidermis offers an excellent opportunity to evaluate the various proposed cellular mechanisms in which centrosome amplification affects tissue development, homeostasis, and tumorigenesis. During embryogenesis, it begins as a single layer of proliferative progenitors, which divide laterally to accommodate embryonic growth, and also perpendicularly to give rise to a stratified, differentiating tissue (27). Only the innermost basal layer retains progenitor status, which relies on integrin-mediated attachment to an underlying basement membrane rich in extracellular matrix. Perpendicular divisions are asymmetric, involving differential Notch and ciliary signaling for proper morphogenesis (28–30). The epidermis matures shortly before birth, and at this stage proliferative basal cells give rise to spinous, granular, and surface stratum corneum cells, which are sloughed and continually replaced by inner cells differentiating upward to maintain homeostasis. In the adult, the epidermis is exposed to a variety of environmental assaults and must undergo frequent turnover to maintain the body’s protective barrier. These features contribute to the skin accounting for the most common cancers worldwide.In the present work, we evaluated centrosome dysfunction in the context of centrosome amplification in mouse epidermis. To do so, we generated mice that induce PLK4 overexpression in the basal layer. PLK4 is the key to initiating centriole duplication, and at elevated levels, this kinase is capable of replicating more than a single centriole on the existing one (31–34). After establishing that the epidermis acquires an excess of centrosomes, we examined the consequences on growth and differentiation, cilia and Notch signaling, mitotic spindle formation and orientation, mitotic error-induced DNA damage response/aneuploidy, and p53-mediated apoptosis. Finally, because our mice remained viable, we could evaluate the effects of sustained centrosome amplification on tissue integrity. Our findings reveal a remarkable resilience of the skin epidermis in coping with abnormalities induced by centrosome amplification. Moreover, despite the longevity of animals that overexpress PLK4 in the skin epidermis, this did not lead to an increased propensity of these mice to initiate or promote tumorigenesis in the skin.     

Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo

Centrosomes are the microtubule-organizing centers of animal cells that organize interphase microtubules and mitotic spindles. Centrioles are the microtubule-based structures that organize centrosomes, and a defined set of proteins, including spindle assembly defective-4 (SAS4) (CPAP/CENPJ), is required for centriole biogenesis. The biological functions of centrioles and centrosomes vary among animals, and the functions of mammalian centrosomes have not been genetically defined. Here we use a null mutation in mouse Sas4 to define the cellular and developmental functions of mammalian centrioles in vivo. Sas4-null embryos lack centrosomes but survive until midgestation. As expected, Sas4^−/− mutants lack primary cilia and therefore cannot respond to Hedgehog signals, but other developmental signaling pathways are normal in the mutants. Unlike mutants that lack cilia, Sas4^−/− embryos show widespread apoptosis associated with global elevated expression of p53. Cell death is rescued in Sas4^−/− p53^−/− double-mutant embryos, demonstrating that mammalian centrioles prevent activation of a p53-dependent apoptotic pathway. Expression of p53 is not activated by abnormalities in bipolar spindle organization, chromosome segregation, cell-cycle profile, or DNA damage response, which are normal in Sas4^−/− mutants. Instead, live imaging shows that the duration of prometaphase is prolonged in the mutants while two acentriolar spindle poles are assembled. Independent experiments show that prolonging spindle assembly is sufficient to trigger p53-dependent apoptosis. We conclude that a short delay in the prometaphase caused by the absence of centrioles activates a previously undescribed p53-dependent cell death pathway in the rapidly dividing cells of the mouse embryo.Centrioles are cylinders of triplet microtubules that provide the template for cilia and nucleate the centrosomes that act as microtubule organizing centers (MTOCs) at spindle poles and during interphase (1, 2). Genetic analysis has demonstrated that the biological roles of centrioles differ widely among organisms: Caenorhabditis elegans embryos without centrioles arrest at the two-cell stage, whereas zygotic removal of centrioles in Drosophila allows survival to adult stages (3–5). In humans, mutations in centriolar and centrosomal proteins are associated with microcephaly or microcephaly in the context of dwarfism (6–10). Abnormal numbers of centrioles are associated with cancer, although it is not clear whether abnormal centrosome number is a cause or an effect of tumorigenesis (1, 11–13). Studies in cultured cell lines have given conflicting results on the roles of vertebrate centrioles in mitosis, chromosome segregation, DNA damage response, and intercellular signaling (14–19), but the precise functions of mammalian centrioles have not been defined genetically.A small number of core proteins have been shown to be required for centriole biogenesis in organisms ranging from Chlamydomonas reinhardtii to human cells. Spindle assembly defective-4 (SAS4), one of these core proteins, acts at an early step in the assembly pathway, when it is required for the addition of tubulin subunits to the forming procentrioles; it also is required for recruitment of the pericentriolar material (PCM) to form the centrosome (3, 20, 21). Mutations in Sas4 block centriole formation in Drosophila and C. elegans, and mutations in human SAS4 (CPAP/CENPJ) cause Seckel syndrome (dwarfism with microcephaly) (3–6). siRNA knockdown of SAS4 in cultured mammalian cells was reported to cause formation of multipolar spindles (14).Here we use null mutations in Sas4 to define the cellular and developmental functions of centrioles in the mouse embryo. As expected, Sas4 is essential for formation of centrioles, centrosomes, and cilia and for cilia-dependent Hedgehog (Hh) signaling. Unexpectedly, Sas4^−/− embryos arrest at an earlier stage than mutants that lack cilia and show widespread cell death associated with strong up-regulation of p53 in most cells in the embryo. Genetic removal of p53 rescues both the cell death and the early lethality of Sas4^−/− mutants. Cell death in the mutants is not associated with defects in the cell-cycle profile, DNA damage response, or chromosome segregation. The data indicate that in Sas4^−/− mouse embryos prolonged prometaphase, caused by a delay in spindle pole assembly, triggers a previously uncharacterized checkpoint that activates p53-dependent apoptosis in vivo.     

Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells

Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. Here, we investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. All 197 genes also produced RNAi phenotypes when centrosomes were present, indicating that none were entirely selective for the acentrosomal pathway. However, a subset of genes produced a selective defect in pole focusing when centrosomes were absent, suggesting that centrosomes compensate for this shape defect. Another subset of genes was specifically associated with the formation of multipolar spindles only when centrosomes were present. We further show that the chromosomal passenger complex orchestrates multiple centrosome-independent processes required for mitotic spindle assembly/maintenance. On the other hand, despite the formation of a chromosome-enriched RanGTP gradient, S2 cells depleted of RCC1, the guanine-nucleotide exchange factor for Ran on chromosomes, established functional bipolar spindles. Finally, we show that cells without functional centrosomes have a delay in chromosome congression and anaphase onset, which can be explained by the lack of polar ejection forces. Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells.Chromosome segregation during mitosis/meiosis is mediated by a microtubule (MT)-based bipolar spindle structure. Mitotic spindle assembly in animal somatic cells was initially believed to rely exclusively on the presence of centrosomes, but it is now well established that centrosomes are not essential (1–6). Land plants and many animal oocytes are paradigmatic examples in which an MT-based spindle normally assembles without centrosomes (7, 8). More recently, it was shown that spindle assembly during somatic cell divisions in the early mouse embryo is also independent of centrosomes (9) and that centrosomes are fully dispensable in planarians throughout their development (10). Overall, these data support the existence of centrosome-independent mechanisms that mediate mitotic/meiotic spindle assembly in animals.Acentrosomal spindle assembly has been particularly well characterized in Xenopus laevis egg extracts, in which MTs form in the vicinity of mitotic chromatin due to a stabilizing effect imposed by a Ras-related nuclear protein in the GTP-bound state (RanGTP) gradient. RanGTP is present at highest concentrations around chromosomes, due to the localization of the Ran guanine nucleotide exchange factor regulator of chromosome condensation 1 (RCC1) on chromosomes (11). However, it remains controversial whether the gradient of RanGTP is required for spindle assembly in other systems (12, 13). Some of the downstream effectors of RanGTP include TPX2 and augmin, which promote MT assembly (14, 15). The chromosomal passenger complex (CPC) has also been implicated in acentrosomal spindle assembly/function in X. laevis egg extracts, as well as in Drosophila and mouse oocytes, and is believed to function independent of RanGTP (16–20). However, despite significant recent progress, a full picture of the molecular mechanisms behind acentrosomal spindle assembly in animal somatic cells is lacking. Moreover, it remains unknown whether the genes involved in acentrosomal spindle assembly are just a subset of those required when centrosomes are present or include specific genes that become essential only when centrosomes are compromised/absent.Here, we investigated the gene requirements for acentrosomal spindle assembly in Drosophila S2 cells by performing a whole-genome RNAi screen. We found that virtually the same set of genes is involved in spindle assembly either with or without centrosomes, although a small subset of genes exhibit a different specific phenotype in the presence or absence of centrosomes.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.Glioblastoma multiforme (GBM) is one of the most aggressive human cancers, and the afflicted patients inevitably succumb. The dismal outcome of this malignancy demands great efforts to find improved methods of treatment (1). Many compounds have been synthesized in our laboratory in the past few years that have proven to be effective against diverse malignant tumors (2–14). These are peptide analogs of hypothalamic hormones: luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), somatostatin, and analogs of other neuropeptides such as bombesin and gastrin-releasing peptide. The receptors for these peptides have been found to be widely distributed in the human body, including in many types of cancers (2–14). The regulatory functions of these hypothalamic hormones and other neuropeptides are not confined to the hypothalamo–hypophyseal system or, even more broadly, to the central nervous system (CNS). In particular, GHRH can induce the differentiation of ovarian granulosa cells and other cells in the reproductive system and function as a growth factor in various normal tissues, benign tumors, and malignancies (2–4, 6, 11, 14–18). Previously, we also reported that antagonistic cytototoxic derivatives of some of these neuropeptides are able to inhibit the growth of several malignant cell lines (2–14).Our earlier studies showed that treatment with antagonists of LHRH or GHRH rarely effects complete regression of glioblastoma-derived tumors (5, 7, 10, 11). Previous studies also suggested that growth factors such as EGF or agonistic analogs of LHRH serving as carriers for cytotoxic analogs and functioning as growth factors may sensitize cancer cells to cytotoxic treatments (10, 19) through the activation of maturation processes. We therefore hypothesized that pretreatment with one of our GHRH agonists, such as JI-34 (20), which has shown effects on growth and differentiation in other cell lines (17, 18, 21, 22), might decrease the pluripotency and the adaptability of GBM cells and thereby increase their susceptibility to cytotoxic treatment.In vivo, tumor cells were implanted into athymic nude mice, tumor growth was recorded weekly, and final tumor mass was measured upon autopsy. In vitro, proliferation assays were used for the determination of neoplastic proliferation and cell growth. Changes in stem (nestin) and maturation (GFAP) antigen expression was evaluated with Western blot studies in vivo and with immunocytochemistry in vitro. The production of glial growth factors (FGF basic, TGFβ) was verified by ELISA. Further, using the Human Cancer Pathway Finder real-time quantitative PCR, numerous genes that play a role in the development of cancer were evaluated. We placed particular emphasis on the measurement of apoptosis, using the ApoLive-Glo Multiplex Assay kit and by detection of the expression of the proapoptotic p53 protein. This overall approach permitted the evaluation of the effect of GHRH agonist, JI-34, on the response to chemotherapy with doxorubicin.     

Spontaneous slow replication fork progression elicits mitosis alterations in homologous recombination-deficient mammalian cells

Homologous recombination deficient (HR⁻) mammalian cells spontaneously display reduced replication fork (RF) movement and mitotic extra centrosomes. We show here that these cells present a complex mitotic phenotype, including prolonged metaphase arrest, anaphase bridges, and multipolar segregations. We then asked whether the replication and the mitotic phenotypes are interdependent. First, we determined low doses of hydroxyurea that did not affect the cell cycle distribution or activate CHK1 phosphorylation but did slow the replication fork movement of wild-type cells to the same level than in HR⁻ cells. Remarkably, these low hydroxyurea doses generated the same mitotic defects (and to the same extent) in wild-type cells as observed in unchallenged HR⁻ cells. Reciprocally, supplying nucleotide precursors to HR⁻ cells suppressed both their replication deceleration and mitotic extra centrosome phenotypes. Therefore, subtle replication stress that escapes to surveillance pathways and, thus, fails to prevent cells from entering mitosis alters metaphase progression and centrosome number, resulting in multipolar mitosis. Importantly, multipolar mitosis results in global unbalanced chromosome segregation involving the whole genome, even fully replicated chromosomes. These data highlight the cross-talk between chromosome replication and segregation, and the importance of HR at the interface of these two processes for protection against general genome instability.DNA is continuously subjected to injury by exogenous and endogenous sources. The faithful transmission of genetic material relies on the DNA damage response (DDR), which coordinates a network of pathways, including DNA replication-repair-recombination, the cell cycle checkpoint, and chromosome segregation. A defect in any of these pathways causes genetic instability and cancer predisposition. Strikingly, both spontaneous DDR activation as a consequence of endogenous replication stress and centrosome abnormalities, which cause uneven chromosome segregation, have been reported in precancerous and early-stage malignancies (1–10). Therefore, endogenous stresses must play a key role in spontaneous chromosome instability and in cancer etiology.Homologous recombination (HR) is an evolutionarily conserved process that controls the balance between genetic stability and diversity. Specifically, HR plays a pivotal role in the reactivation of replication forks that have been blocked, contributing to DNA replication accuracy (11–16). Replication forks are routinely inactivated by endogenous stress (17, 18); therefore, HR should play an essential role to protect cells against these types of stresses, and HR deficiency should reveal endogenous replication stress. Remarkably, unchallenged HR-deficient (HR⁻) cells display both a genome-wide decrease in replication fork speed (19) and a spontaneous increase in the frequency of cells containing extra centrosomes (20–28). Two hypotheses may account for these two phenotypes. First, replication stress leads to chromosome alteration at incomplete replicated regions and chromosome rearrangements (29). However, centrosomes do not contain DNA, and if extra centrosomes at mitosis [mitotic extra centrosome (MEC)] are active, unbalanced chromosome segregation should lead to global chromosome instability, even for fully replicated chromosomes. Second, HR proteins are associated with supernumerary centrosomes; therefore, centrosome duplication defects may directly result from HR misregulation (30, 31).In this study, we addressed whether spontaneous MECs result from slow replication fork movement in HR⁻ cells. The data presented here underline the importance of HR at the molecular interface between replication and chromosome segregation to protect against spontaneous genomic instability.     

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.Cell division is a fundamental biological process that is tightly regulated by evolutionarily conserved signaling pathways (1, 2). The initial decision to start cell division, the fidelity of subsequent DNA replication, and the final formation of daughter cells is monitored and regulated by these essential pathways (2–6). The cyclin-dependent kinases (CDKs) are the central players that orchestrate this orderly progression through the cell cycle (1, 2, 6, 7). The enzymatic activity of CDKs is regulated by complex mechanisms that include posttranslational modifications and expression of activating and inhibitory proteins (1, 2, 6, 7). The spatial and temporal changes in the activity of these CDK complexes are thought to generate the distinct substrate specificities that lead to sequential and unidirectional progression of the cell cycle (1, 8, 9).Cell-cycle deregulation is a universal feature of human cancer and a long-sought-after target for anticancer therapy (1, 10–13). Frequent genetic or epigenetic changes in mitogenic pathways, CDKs, cyclins, or CDK inhibitors are observed in various human cancers (1, 4, 11). In particular, the G1/S-regulating CDK4/6–cyclin D–inhibitors of CDK4 (INK4)–retinoblastoma (Rb) protein pathway frequently is disrupted in cancer cells (11, 14). These observations provided an impetus to develop CDK inhibitors as anticancer drugs. However, the earlier class of CDK inhibitors had limited specificity, inadequate clinical activity, poor pharmacokinetic properties, and unacceptable toxicity profiles (10, 11, 14, 15). These disappointing initial efforts now have been followed by the development of the specific CDK4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219), which have demonstrated manageable toxicities, improved pharmacokinetic properties, and impressive antitumor activity, especially in certain forms of breast cancer (14, 16). Successful early clinical trials with these three CDK4/6 inhibitors have generated cautious enthusiasm that these drugs may emerge as a new class of anticancer agents (14, 17). Palbociclib recently was approved by Food and Drug Administration for the treatment of metastatic breast cancer and became the first CDK4/6 inhibitor approved for anticancer therapy (18).In addition to its potential as an anticancer strategy, CDK4/6 inhibition in normal tissues could be exploited therapeutically for wide-ranging clinical conditions. For example, radiation-induced myelosuppression, caused by cell death of proliferating hematopoietic stem/progenitor cells, can be rescued by palbociclib (19, 20). Furthermore, cytotoxic anticancer agents cause significant toxicities to normal proliferating cells, which possibly could be mitigated by the concomitant use of CDK4/6 inhibitors (20, 21). More broadly, cell-cycle inhibition could have beneficial effects in disorders in which maladaptive proliferation of normal cells contributes to the disease pathology, as observed in vascular proliferative diseases, hyperproliferative skin diseases, and autoimmune disorders (22, 23). In support of this possibility, palbociclib treatment recently was reported to ameliorate disease progression in animal models of rheumatoid arthritis through cell-cycle inhibition of synovial fibroblasts (24).Abnormal cellular proliferation also is a hallmark of various kidney diseases (25), and cell-cycle inhibition has been shown to ameliorate significantly the pathogenesis of polycystic kidney disease (26), nephritis (27), and acute kidney injury (AKI) (28). Remarkably, during AKI, the normally quiescent renal tubular cells reenter the cell cycle (29–34), and blocking cell-cycle progression can reduce renal injury (28). Here, we provide evidence that the CDK4/6 pathway is activated early during AKI and demonstrate significant protective effects of CDK4/6 inhibitors in animal models of cisplatin-induced AKI. In addition, we found that the CDK4/6 inhibitors palbociclib and LEE011 are potent inhibitors of organic cation transporter 2 (OCT2), a cisplatin uptake transporter highly expressed in renal tubular cells (35–37). Our findings provide a rationale for the clinical development of palbociclib and LEE011 for the prevention and treatment of AKI.     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.     

Transition paths,diffusive processes,and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.Protein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1, 2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7–15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16–25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the free energy minimum of the unfolded state. The development of ultrafast laser spectroscopy (29, 30) and single-molecule (27, 28, 31) techniques have made it possible to establish upper bounds on the transition path time (t_TP) ranging from <200 and <10 μs by earlier (27) and more recent (28), respectively, direct single-molecule FRET to <2 μs (30) by bulk relaxation measurements. Consistent with these observations, recent extensive atomic simulations have also provided estimated t_TP values of the order of ∼1 μs (32, 33). These advances offer exciting prospects of characterizing the productive events along folding TPs.It is timely, therefore, to further the theoretical investigation of TP-related questions (19). To this end, we used coarse-grained C_α models (14) to perform extensive simulations of the folding trajectories of small proteins with 56- to 86-aa residues. These tractable models are useful, because despite significant progress, current atomic models cannot provide the same degree of sampling coverage for proteins of comparable sizes (32, 33). In addition to structural insights, this study provides previously unexplored vantage points to compare the diffusion and TST pictures of folding. Deviations of folding behaviors from TST predictions are not unexpected, because TST is mostly applicable to simple gas reactions; however, the nature and extent of the deviations have not been much explored. Our explicit-chain simulation data conform well to the diffusion picture but not as well to TST. In particular, the preexponential factors of the simulated folding rates exhibit a small but appreciable variation that depends on native topology. These findings and others reported below underscore the importance of single-molecule measurements (13, 27, 28, 31, 34, 35) in assessing the merits of proposed scenarios and organizing principles of folding (7–25, 36, 37).     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.