Inhibition of NF-κB-Mediated Proinflammatory Transcription by Ru(II) Complexes of Anti-Angiogenic Ligands in Triple-Negative Breast Cancer.

Nuclear factor kappa beta (NF-κB) plays a pivotal role in breast cancer, particularly triple-negative breast cancer, by promoting inflammation, proliferation, epithelial-mesenchymal transition, metastasis, and drug resistance. Upregulation of NF-κB boosts vascular endothelial growth factor (VEGF) expression, assisting angiogenesis. The Ru(II) complexes of methyl- and dimethylpyrazolyl-benzimidazole N,N donors inhibit phosphorylation of ser536 in p65 and translocation of the NF-κB heterodimer (p50/p65) to the nucleus, disabling transcription to upregulate inflammatory signaling. The methyl- and dimethylpyrazolyl-benzimidazole inhibit VEGFR2 phosphorylation at Y1175, disrupting downstream signaling through PLC-γ and ERK1/2, ultimately suppressing Ca(II)-signaling. Partial release of the antiangiogenic ligand in a reactive oxygen species-rich environment is possible as per our observation to inhibit both NF-κB and VEGFR2 by the complexes. The complexes are nontoxic to zebrafish embryos up to 50 µM, but the ligands show strong in vivo antiangiogenic activity at 3 µM during embryonic growth in Tg(fli1:GFP) zebrafish but no visible effect on the adult phase.

Neuroendocrine and cellular mechanisms in stress resilience: From hormonal influence in the CNS to mitochondrial dysfunction and oxidative stress.

Recent advancements in neuroendocrinology challenge the long-held belief that hormonal effects are confined to perivascular tissues and do not extend to the central nervous system (CNS). This paradigm shift, propelled by groundbreaking research, reveals that synthetic hormones, notably in anti-inflammatory medications, significantly influence steroid psychosis, behavioural, and cognitive impairments, as well as neuropeptide functions. A seminal development in this field occurred in 1968 with McEven's proposal that rodent brains are responsive to glucocorticoids, fundamentally altering the understanding of how anxiety impacts CNS functionality and leading to the identification of glucocorticosteroids and mineralocorticoids as distinct corticotropic receptors. This paper focuses on the intricate roles of the neuroendocrine, immunological, and CNS in fostering stress resilience, underscored by recent animal model studies. These studies highlight active, compensatory, and passive strategies for resilience, supporting the concept that anxiety and depression are systemic disorders involving dysregulation across both peripheral and central systems. Resilience is conceptualized as a multifaceted process that enhances psychological adaptability to stress through adaptive mechanisms within the immunological system, brain, hypothalamo-pituitary-adrenal axis, and ANS Axis. Furthermore, the paper explores oxidative stress, particularly its origin from the production of reactive oxygen species (ROS) in mitochondria. The mitochondria's role extends beyond ATP production, encompassing lipid, heme, purine, and steroidogenesis synthesis. ROS-induced damage to biomolecules can lead to significant mitochondrial dysfunction and cell apoptosis, emphasizing the critical nature of mitochondrial health in overall cellular function and stress resilience. This comprehensive synthesis of neuroendocrinological and cellular biological research offers new insights into the systemic complexity of stress-related disorders and the imperative for multidisciplinary approaches in their study and treatment.

Molecular basis of VEGFR1 autoinhibition at the plasma membrane.

Ligand-independent activation of VEGFRs is a hallmark of diabetes and several cancers. Like EGFR, VEGFR2 is activated spontaneously at high receptor concentrations. VEGFR1, on the other hand, remains constitutively inactive in the unligated state, making it an exception among VEGFRs. Ligand stimulation transiently phosphorylates VEGFR1 and induces weak kinase activation in endothelial cells. Recent studies, however, suggest that VEGFR1 signaling is indispensable in regulating various physiological or pathological events. The reason why VEGFR1 is regulated differently from other VEGFRs remains unknown. Here, we elucidate a mechanism of juxtamembrane inhibition that shifts the equilibrium of VEGFR1 towards the inactive state, rendering it an inefficient kinase. The juxtamembrane inhibition of VEGFR1 suppresses its basal phosphorylation even at high receptor concentrations and transiently stabilizes tyrosine phosphorylation after ligand stimulation. We conclude that a subtle imbalance in phosphatase activation or removing juxtamembrane inhibition is sufficient to induce ligand-independent activation of VEGFR1 and sustain tyrosine phosphorylation.

SAXS/MC studies of the mixed-folded protein Cdt1 reveal monomeric, folded over conformations.

Cdt1 is a protein critical for DNA replication licensing and is well-established to be a binding partner of the minichromosome maintenance (MCM) complex. Cdt1 has also been demonstrated to have an emerging, "moonlighting" role at the kinetochore via direct binding to microtubules and to the Ndc80 complex. However, it is not known how the structure and conformations of Cdt1 could allow for these multiple, completely unique sets of protein complexes. And while there exist multiple robust methods to study entirely folded or entirely unfolded proteins, structure-function studies of combined, mixed folded/disordered proteins remain challenging. It this work, we employ multiple orthogonal biophysical and computational techniques to provide a detailed structural characterization of human Cdt1 92-546. DSF and DSCD show both folded winged helix (WH) domains of Cdt1 are relatively unstable. CD and NMR show the N-terminal and the linker regions are intrinsically disordered. Using DLS and SEC-MALS, we show that Cdt1 is polydisperse, monomeric at high concentrations, and without any apparent inter-molecular self-association. SEC-SAXS of the monomer in solution enabled computational modeling of the protein in silico. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble. Using experimental SAXS data, we filtered for conformations which did and did not fit our data. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with our SAXS data. The best fit models have the N-terminal and linker regions extended into solution and the two folded domains close to each other in apparent "folded over" conformations. The best fit Cdt1 conformations are consistent with a function as a scaffold protein which may be sterically blocked without the presence of binding partners. Our studies also provide a template for combining experimental and computational biophysical techniques to study mixed-folded proteins.

Nuclear lamin A-associated proteins are required for centromere assembly.

Many Lamin A-associated proteins (LAAP's) that are key constituents of the nuclear envelope (NE), assemble at the "core" domains of chromosomes during NE reformation and mitotic exit. However, the identity and function of the chromosomal core domains remain ill-defined. Here, we show that a distinct section of the core domain overlaps with the centromeres/kinetochores of chromosomes during mitotic telophase. The core domain can thus be demarcated into a kinetochore proximal core (KPC) on one side of the segregated chromosomes and the kinetochore distal core (KDC) on the opposite side, close to the central spindle. We next tested if centromere assembly is connected to NE re-formation. We find that centromere assembly is markedly perturbed after inhibiting the function of LMNA and the core-localized LAAPs, BANF1 and Emerin. We also find that the LAAPs exhibit multiple biochemical interactions with the centromere and inner kinetochore proteins. Consistent with this, normal mitotic progression and chromosome segregation was severely impeded after inhibiting LAAP function. Intriguingly, the inhibition of centromere function also interferes with the assembly of LAAP components at the core domain, suggesting a mutual dependence of LAAP and centromeres for their assembly at the core domains. Finally, we find that the localization of key proteins involved in the centromeric loading of CENP-A, including the Mis18 complex and HJURP were markedly affected in LAAP-inhibited cells. Our evidence points to a model where LAAP assembly at the core domain serves a key function in loading new copies of centromeric proteins during or immediately after mitotic exit.

The Ndc80-Cdt1-Ska1 complex is a central processive kinetochore-microtubule coupling unit.

It is known that microtubule-binding proteins including the Ska1 complex and the DNA replication licensing factor, Cdt1, enable the kinetochore-localized Ndc80 complex to form robust kinetochore-microtubule attachments. However, it is not clear how the Ndc80 complex is stably coupled to dynamic spindle microtubule plus-ends. Here, we have developed a conditional auxin-inducible degron approach to reveal a function for Cdt1 in chromosome segregation and kinetochore-microtubule interactions that is separable from its role in DNA replication licensing. Further, we demonstrate that a direct interaction between Cdt1 and Ska1 is required for recruiting Cdt1 to kinetochores and spindle microtubules. Cdt1 phosphorylation by Cdk1 kinase is critical for Ska1 binding, kinetochore-microtubule attachments, and mitotic progression. Furthermore, we show that Cdt1 synergizes with Ndc80 and Ska1 for microtubule binding, including forming a diffusive, tripartite Ndc80-Cdt1-Ska1 complex that can processively track dynamic microtubule plus-ends in vitro. Taken together, our data identify the Ndc80-Cdt1-Ska1 complex as a central molecular unit that can promote processive bidirectional tip-tracking of microtubules by kinetochores.

Coordination between the Ndc80 complex and dynein is essential for microtubule plus-end capture by kinetochores during early mitosis.

Mitotic kinetochores are initially captured by dynamic microtubules via a "search-and-capture" mechanism. The microtubule motor, dynein, is critical for kinetochore capture as it has been shown to transport microtubule-attached chromosomes toward the spindle pole during prometaphase. The microtubule-binding nuclear division cycle 80 (Ndc80) complex that is recruited to kinetochores in prophase is known to play a central role in forming kinetochore-microtubule (kMT) attachments in metaphase. It is not yet clear, however, how Ndc80 contributes to initial kMT capture during prometaphase. Here, by combining CRISPR/Cas9-mediated knockout and RNAi technology with assays specific to study kMT capture, we show that mitotic cells lacking Ndc80 exhibit substantial defects in this function during prometaphase. Rescue experiments show that Ndc80 mutants deficient in microtubule-binding are unable to execute proper kMT capture. While cells inhibited of dynein alone are predominantly able to make initial kMT attachments, cells co-depleted of Ndc80 and dynein show severe defects in kMT capture. Further, we use an in vitro total internal reflection fluorescence microscopy assay to reconstitute microtubule capture events, which suggest that Ndc80 and dynein coordinate with each other for microtubule plus-end capture and that the phosphorylation status of Ndc80 is critical for productive kMT capture. A novel interaction between Ndc80 and dynein that we identify in prometaphase extracts might be critical for efficient plus-end capture. Thus, our studies, for the first time, identify a distinct event in the formation of initial kMT attachments, which is directly mediated by Ndc80 and in coordination with dynein is required for efficient kMT capture and chromosome alignment.

CLASP2 recognizes tubulins exposed at the microtubule plus-end in a nucleotide state-sensitive manner.

CLASPs (cytoplasmic linker-associated proteins) are ubiquitous stabilizers of microtubule dynamics, but their molecular targets at the microtubule plus-end are not understood. Using DNA origami-based reconstructions, we show that clusters of human CLASP2 form a load-bearing bond with terminal non-GTP tubulins at the stabilized microtubule tip. This activity relies on the unconventional TOG2 domain of CLASP2, which releases its high-affinity bond with non-GTP dimers upon their conversion into polymerization-competent GTP-tubulins. The ability of CLASP2 to recognize nucleotide-specific tubulin conformation and stabilize the catastrophe-promoting non-GTP tubulins intertwines with the previously underappreciated exchange between GDP and GTP at terminal tubulins. We propose that TOG2-dependent stabilization of sporadically occurring non-GTP tubulins represents a distinct molecular mechanism to suppress catastrophe at the freely assembling microtubule ends and to promote persistent tubulin assembly at the load-bearing tethered ends, such as at the kinetochores in dividing cells.

Biocompatible and optically stable hydrophobic fluorescent carbon dots for isolation and imaging of lipid rafts in model membrane.

Lateral heterogeneity in cell membranes features a variety of compositions that influence their inherent properties. One such biophysical variation is the formation of a membrane or lipid raft, which plays important roles in many cellular processes. The lipid rafts on the cell membrane are mostly identified by specific dyes and heavy metal quantum dots, which have their own drawbacks, such as cytotoxicity, photostability, and incompatibility. To this end, we synthesized special, hydrophobic, fluorescent, photostable, and non-cytotoxic carbon dots (CDs) by solvent-free thermal treatment using non-cytotoxic materials and incorporated into the lipid bilayers of giant unilamellar vesicles (GUVs) made from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) lipids. A 2:2:1 mixture of DOPC, DPPC, and cholesterol (Chol) develops lipid rafts on the membrane by phase separation. The photophysical properties of the CDs get modulated on incorporation into the lipid rafts that identifies the membrane heterogeneity. The main attempt in this work is to develop a new, simple, cost-effective, and bio-friendly lipid raft marker, which can be used in biological applications, alongside other conventional raft markers, with more advantages.

In Vitro and In Vivo Approaches to Study Kinetochore-Microtubule Attachments During Mitosis.

The separation of duplicated chromosomes during mitosis is a pivotal step in the process of cellular division. Therefore, the orchestrated events that take place to ensure proper attachment and stabilization of kMTs are keen areas of interest in the mitosis field. Here we describe the methods used to study kMT attachments via in vitro biochemical methods and in vivo cell biological approaches.

Cytotoxic Ruthenium(II) Complexes of Pyrazolylbenzimidazole Ligands That Inhibit VEGFR2 Phosphorylation.

Eight new ruthenium(II) complexes of N,N-chelating pyrazolylbenzimidazole ligands of the general formula [RuII(p-cym)(L)X]+ [where the ligand L is 2-(1H-pyrazol-1-yl)-1H-benzo[d]imidazole (L1) substituted at the 4 position of the pyrazole ring by Cl (L2), Br (L3), or I (L4) and X = Cl- and I-] were synthesized and characterized using various analytical techniques. Complexes 1 and 3 were also characterized by single-crystal X-ray crystallography, and they crystallized as a monoclinic crystal system in space groups P21/n and P21/c, respectively. The complexes display good solution stability at physiological pH 7.4. The iodido-coordinated pyrazolylbenzimidazole ruthenium(II) p-cymene complexes (2, 4, 6, and 8) are more resistant toward hydrolysis and have less tendency to form monoaquated complexes in comparison to their chlorido analogues (1, 3, 5, and 7). The halido-substituted 2-(1H-pyrazol-1-yl)-1H-benzo[d]imidazole ligands, designed as organic-directing molecules, inhibit vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation. In addition, the ruthenium(II) complexes display a potential to bind to DNA bases. The cytotoxicity profile of the complexes (IC50 ca. 9-12 µM for 4-8) against the triple-negative breast cancer cells (MDA-MB-231) show that most of the complexes are efficient. The lipophilicity and cellular accumulation data of the complexes show a good correlation with the cytotoxicity profile of 1-8. The representative complexes 3 and 7 demonstrate the capability of arresting the cell cycle in the G2/M phase and induce apoptosis. The inhibition of VEGFR2 phosphorylation with the representative ligands L2 and L4 and the corresponding metal complexes 3 and 7 in vitro shows that the organic-directing ligands and their complexes inhibit VEGFR2 phosphorylation. Besides, L2, L4, 3, and 7 inhibit the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and proto-oncogene tyrosine-protein kinase (Src), capable of acting downstream of VEGFR2 as well as independently. Compounds L2, L4, 3, and 7 have a lesser effect on ERK1/2 and more prominently affect Src phosphorylation. We extended the study for L2 and 3 in the Tg(fli1:gfp) zebrafish model and found that L2 is more effective in vivo compared to 3 in inhibiting angiogenesis.

Hypoxia Active Platinum(IV) Prodrugs of Orotic Acid Selective to Liver Cancer Cells.

Platinum(IV) complexes of orotic acid selectively target liver cancer cells displaying enhanced activity and higher uptake in Hep G2. The comparatively higher expression of Organic Anion Transporter 2 (OAT2) in Hep G2 and decrease in toxicity in the presence of OAT2 inhibitor suggest its involvement in the uptake of the complexes. They are resistant to sequestration by the copper transporter ATP7B, unlike cisplatin and oxaliplatin.

Selective targeting of the inactive state of hematopoietic cell kinase (Hck) with a stable curcumin derivative.

Hck, a Src family nonreceptor tyrosine kinase (SFK), has recently been established as an attractive pharmacological target to improve pulmonary function in COVID-19 patients. Hck inhibitors are also well known for their regulatory role in various malignancies and autoimmune diseases. Curcumin has been previously identified as an excellent DYRK-2 inhibitor, but curcumin's fate is tainted by its instability in the cellular environment. Besides, small molecules targeting the inactive states of a kinase are desirable to reduce promiscuity. Here, we show that functionalization of the 4-arylidene position of the fluorescent curcumin scaffold with an aryl nitrogen mustard provides a stable Hck inhibitor (Kd = 50 ± 10 nM). The mustard curcumin derivative preferentially interacts with the inactive conformation of Hck, similar to type-II kinase inhibitors that are less promiscuous. Moreover, the lead compound showed no inhibitory effect on three other kinases (DYRK2, Src, and Abl). We demonstrate that the cytotoxicity may be mediated via inhibition of the SFK signaling pathway in triple-negative breast cancer and murine macrophage cells. Our data suggest that curcumin is a modifiable fluorescent scaffold to develop selective kinase inhibitors by remodeling its target affinity and cellular stability.

Disruption of the Microtubule Network and Inhibition of VEGFR2 Phosphorylation by Cytotoxic N,O-Coordinated Pt(II) and Ru(II) Complexes of Trimethoxy Aniline-Based Schiff Bases.

Platinum-based complexes are one of the most successful chemotherapeutic agents having a significant ground in cancer chemotherapy despite their side effects. During the past few decades, Ru(II) complexes have been emerging as efficient alternatives owing to their promising activities against platinum-resistant cancer. The pathway of action, lipophilicity, and cytotoxicity of a Pt or Ru complex may be tuned by varying the attached ligands, the coordination mode, and the leaving group. In this work, we report a family of Pt(II) and Ru(II) complexes (1-5) of three N,O and N,N donor-based trimethoxyanilines containing Schiff bases with the general formula [PtII(L)(DMSO)Cl], [RuII(L)(p-cymene)Cl], [RuII(L)(p-cymene)Cl]+, and [PtII(L)Cl2]. All of the complexes are characterized by different analytical techniques. 1H NMR and electrospray ionization mass spectrometry (ESI-MS) data suggest that the N,O-coordinated Pt(II) complexes undergo slower aquation compared to the Ru(II) analogues. The change of the coordination mode to N,N causes the Ru complexes to be more inert to aquation. The N,O-coordinating complexes show superiority over N,N-coordinating complexes by displaying excellent in vitro antiproliferative activity against different aggressive cancer cells, viz., triple-negative human metastatic breast adenocarcinoma MDA-MB-231, human pancreatic carcinoma MIA PaCa-2, and hepatocellular carcinoma Hep G2. In vitro cytotoxicity studies suggest that Pt(II) complexes are more effective than their corresponding Ru(II) analogues, and the most cytotoxic complex 3 is 10-15 times more toxic than the clinical drugs cisplatin and oxaliplatin against MDA-MB-231 cells. Cellular studies show that all of the N,O-coordinated complexes (1-3) initiate disruption of the microtubule network in MDA-MB-231 cells in a dose-dependent manner within 6 h of incubation and finally lead to the arrest of the cell cycle in the G2/M phase and render apoptotic cell death. The disruption of the microtubule network affects the agility of the cytoskeleton rendering inhibition of tyrosine phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2), a key step in angiogenesis. Complexes 1 and 2 inhibit VEGFR2 phosphorylation in a dose-dependent fashion. Among the Pt(II) and Ru(II) complexes, the former displays higher cytotoxicity, a stronger effect on the cytoskeleton, better VEGFR2 inhibition, and strong interaction with the model nucleobase 9-ethylguanine (9-EtG).

Kinetochore-microtubule coupling mechanisms mediated by the Ska1 complex and Cdt1.

The faithful segregation of duplicated sister chromatids rely on the remarkable ability of kinetochores to sustain stable load bearing attachments with the dynamic plus ends of kinetochore-microtubules (kMTs). The outer layer of the kinetochore recruits several motor and non-motor microtubule-associated proteins (MAPs) that help the kinetochores establish and maintain a load bearing dynamic attachment with kMTs. The primary kMT-binding protein, the Ndc80 complex (Ndc80c), which is highly conserved among diverse organisms from yeast to humans, performs this essential function with assistance from other MAPs. These MAPs are not an integral part of the kinetochore, but they localize to the kinetochore periodically throughout mitosis and regulate the strength of the kinetochore microtubule attachments. Here, we attempt to summarize the recent advances that have been made toward furthering our understanding of this co-operation between the Ndc80c and these MAPs, focusing on the spindle and kinetochore-associated 1 (Ska1) complex (Ska1c) and Cdc10-dependent transcript 1 (Cdt1) in humans.

Sirtuins family as a target in endothelial cell dysfunction: implications for vascular ageing.

The vascular endothelium is a protective barrier between the bloodstream and the vasculature that may be disrupted by different factors such as the presence of diseased states. Diseases like diabetes and obesity pose a great risk toward endothelial cell inflammation and oxidative stress, leading to endothelial cell dysfunction and thereby cardiovascular complications such as atherosclerosis. Sirtuins are NAD+-dependent histone deacetylases that are implicated in the pathophysiology of cardiovascular diseases, and they have been identified to be important regulators of endothelial cell function. A handful of recent studies suggest that disbalance in the regulation of endothelial sirtuins, mainly sirtuin 1 (SIRT1), contributes to endothelial cell dysfunction. Herein, we summarize how SIRT1 and other sirtuins may contribute to endothelial cell function and how presence of diseased conditions may alter their expressions to cause endothelial dysfunction. Moreover, we discuss how the beneficial effects of exercise on the endothelium are dependent on SIRT1. These mainly include regulation of signaling pathways related to endothelial nitric oxide synthase phosphorylation and nitric oxide production, mitochondrial biogenesis and mitochondria-mediated apoptotic pathways, oxidative stress and inflammatory pathways. Sirtuins as modulators of the adverse conditions in the endothelium hold a promising therapeutic potential for health conditions related to endothelial dysfunction and vascular ageing.

Microtubule end conversion mediated by motors and diffusing proteins with no intrinsic microtubule end-binding activity.

Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end-directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics.

Endothelial dysfunction in diabetes and hypertension: Role of microRNAs and long non-coding RNAs.

The vascular endothelium acts as a barrier between the blood flow and the inner lining of the vessel wall, and it functions as a filtering machinery to filter out any unwanted transfer of materials from both sides (i.e. the blood and the surrounding tissues). It is evident that diseases such as diabetes, obesity, and hypertension disturb the normal endothelial functions in humans and lead to endothelial dysfunction, which may further precede to the development of atherosclerosis. Long non-coding RNAs and micro RNAs both are types of non-coding RNAs which, in the recent years, have increasingly been studied in the pathophysiology of many diseases including diabetes, obesity, cardiovascular diseases, neurological diseases, and others. Recent findings have pointed out important aspects on their relevance to endothelial function as well as dysfunction of the system which may arise from presence of diseases such as diabetes and hypertension. Diabetes or hypertension-mediated endothelial dysfunction show characteristics such as reduced nitric oxide synthesis through suppression of endothelial nitric oxide synthase activity in endothelial cells, reduced sensitivity of nitric oxide in smooth muscle cells, and inflammation - all of which have been either shown to be directly caused by gene regulatory mechanisms of non-coding RNAs or shown to be having a correlation with them. In this review, we aim to discuss such findings on the role of these non-coding RNAs in diabetes or hypertension-associated endothelial dysfunction and the related mechanisms that may pave the way for alleviating endothelial dysfunction and its related complications such as atherosclerosis.

In vitro reconstitution of lateral to end-on conversion of kinetochore-microtubule attachments.

During mitosis, kinetochores often bind to the walls of spindle microtubules, but these lateral interactions are then converted into a different binding mode in which microtubule plus-ends are embedded at kinetochores, forming dynamic "end-on" attachments. This remarkable configuration allows continuous addition or loss of tubulin subunits from the kinetochore-bound microtubule ends, concomitant with movement of the chromosomes. Here, we describe novel experimental assays for investigating this phenomenon using a well-defined in vitro reconstitution system visualized by fluorescence microscopy. Our assays take advantage of the kinetochore kinesin CENP-E, which assists in microtubule end conversion in vertebrate cells. In the experimental setup, CENP-E is conjugated to coverslip-immobilized microbeads coated with selected kinetochore components, creating conditions suitable for microtubule gliding and formation of either static or dynamic end-on microtubule attachment. This system makes it possible to analyze, in a systematic and rigorous manner, the molecular friction generated by the microtubule wall-binding proteins during lateral transport, as well as the ability of these proteins to establish and maintain association with microtubule plus-end, providing unique insights into the specific activities of various kinetochore components.

ATP and magnesium promote cotton short-form ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase hexamer formation at low micromolar concentrations.

We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton ß-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 µM). Hexamer fractions peak at 30 µM and dominate at 8-70 µM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 µM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 µM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.