Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics

Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that interact and dissociate the tubulin dimer. Here we show how TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO– present in TBCB, which is similar to the EEY/F-COO– element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE–TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated to microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation

Cell Membrane-Coated Nanoparticles for Precision Medicine: A Comprehensive Review of Coating Techniques for Tissue-Specific Therapeutics

Nanoencapsulation has become a recent advancement in drug delivery, enhancing stability, bioavailability, and enabling controlled, targeted substance delivery to specific cells or tissues. However, traditional nanoparticle delivery faces challenges such as a short circulation time and immune recognition. To tackle these issues, cell membrane-coated nanoparticles have been suggested as a practical alternative. The production process involves three main stages: cell lysis and membrane fragmentation, membrane isolation, and nanoparticle coating. Cell membranes are typically fragmented using hypotonic lysis with homogenization or sonication. Subsequent membrane fragments are isolated through multiple centrifugation steps. Coating nanoparticles can be achieved through extrusion, sonication, or a combination of both methods. Notably, this analysis reveals the absence of a universally applicable method for nanoparticle coating, as the three stages differ significantly in their procedures. This review explores current developments and approaches to cell membrane-coated nanoparticles, highlighting their potential as an effective alternative for targeted drug delivery and various therapeutic applications

Isolation of microtubules and microtubule proteins

This unit describes various protocols for the isolation and purification of the main constituents of microtubules, chiefly α- and β-tubulin, and the most significant microtubule associated proteins (MAPs), specifically MAP1A, MAP1B, MAP2, and tau. We include a classical isolation method for soluble tubulin heterodimer as the first basic purification protocol. In addition, we show how to analyze the tubulin and MAPs obtained after a phosphocellulose chromatography purification procedure. This unit also details a powerful and simple method to determine the native state of the purified tubulin based on one-dimensional electrophoresis under nondenaturing conditions (UNIT 6.5). The last protocol describes the application of a new technique that allows visualizing the quality of polymerized microtubules based on atomic force microscopy (AFM).Peer reviewe

Regulated expression of p14 (cofactor A) during spermatogenesis

The correct folding of tubulins and the generation of functional αβ-tubulin heterodimers require the participation of a series of recently described molecular chaperones and CCT (or TRiC), the cytosolic chaperonin containing TCP-1. p14 (cofactor A) is a highly conserved protein that forms stable complexes with β-tubulin which are not apparently indispensable along the in vitro β-tubulin folding route. Consequently, the precise role of p14 is still unknown, though findings on Rbl2p (its yeast homologue) suggest p14 might play a role in meiosis and/or perhaps to serve as an excess β-tubulin reservoir in the cell. This paper investigates the in vivo possible role of p14 in testis where mitosis, meiosis, and intense microtubular remodeling processes occur. Our results confirm that p14 is more abundantly expressed in testis than in other adult mammalian tissues. Northern blot, Western blot, in situ hybridization, and immunocytochemical analyses have all demonstrated that p14 is progressively upregulated from the onset of meiosis through spermiogenesis, being more abundant in differentiating spermatids. The close correlation observed between the mRNA expression waves for p14 and testis specific tubulin isotypes β3 and α3/7, together with the above results, suggest that p14 role in testis would presumably be associated to β-tubulin processing rather than meiosis itself. Additional in vitro β3-tubulin synthesis experiments have shown that p14 plays a double role in β-tubulin folding, enhancing the dimerization of newly synthesized β-tubulin isotypes as well as capturing excess β-tubulin monomers. The above evidence suggests that p14 is a chaperone required for the actual β-tubulin folding process in vivo and storage of excess β-tubulin in situations, such as in testis, where excessive microtubule remodeling could lead to a disruption of the α-β balance. As seen for other chaperones, p14 could also serve as a route to lead excess β-tubulin or replaced isotypes towards degradation.Contract grant sponsor: Caja Cantabria/Universidad de Cantabria; Contract grant numbers: DGICYT (Ministry of Education) PB97–0350, DGICYT PB95–0119, EC (European Commission) PL96–0183, and CAM (Caja ahorros, Madrid) 07/0022.Peer reviewe

Role of cofactors B (TBCB) and E (TBCE) in tubulin heterodimer dissociation

Tubulin folding cofactors B (TBCB) and E (TBCE) are α-tubulin binding proteins that, together with Arl2 and cofactors D (TBCD), A (TBCA or p14) and C (TBCC), participate in tubulin biogenesis. TBCD and TBCE have also been implicated in microtubule dynamics through regulation of tubulin heterodimer dissociation. Understanding the in vivo function of these proteins will shed light on the Kenny–Caffey/Sanjad–Sakati syndrome, an important human disorder associated with TBCE. Here we show that, when overexpressed, TBCB depolymerizes microtubules. We found that this function is based on the ability of TBCB to form a binary complex with TBCE that greatly enhances the efficiency of this cofactor to dissociate tubulin in vivo and in vitro. We also show that TBCE, TBCB and α-tubulin form a ternary complex after heterodimer dissociation, whereas the free β-tubulin subunit is recovered by TBCA. These complexes might serve to escort α-tubulin towards degradation or recycling, depending on the cell requirements.D.K. and G.C. were supported by fellowships from the Universidad de Cantabria and the Fundación Marqués de Valdecilla-IFIMAV, respectively. J.B. and this work were supported by grants from the Spanish Ministry of Science and Technology to J.C.Z. (BMC2001-0618 and BFU2004-01212) and from the Fundación Marqués de Valdecilla-IFIMAV (A/36/01, A/32/03 and API/05/1/8).Peer reviewe

Carbon nanotubes targeted to the tumor microenvironment inhibit metastasis in a preclinical model of melanoma

Despite notable progress in cancer therapy, metastatic diseases continue to be the primary cause of cancer-related mortality. Multi-walled carbon nanotubes (MWCNTs) can enter tissues and cells and interfere with the dynamics of the cytoskeletal nanofilaments biomimetically. This endows them with intrinsic anti-tumoral effects comparable to those of microtubule-binding chemotherapies such as Taxol®.In this study, our focus was on exploring the potential of oxidized MWCNTs in selectively targeting the vascular endothelial growth factor receptor (VEGFR). Our objective was to evaluate their effectiveness in inhibiting metastatic growth by inducing anti-proliferative, anti-migratory, and cytotoxic effects on both cancer and tumor microenvironment cells. Our findings demonstrated a significant reduction of over 80 % in malignant melanoma lung metastases and a substantial enhancement in overall animal welfare following intravenous administration of the targeted biodegradable MWCNTs. Furthermore, the combination of these nanomaterials with the conventional chemotherapy agent Taxol® yielded a remarkable 90 % increase in the antimetastatic effect. These results highlight the promising potential of this combined therapeutic approach against metastatic disease and are of paramount importance as metastasis is responsible for nearly 60,000 deaths each year

The unpredictable carbon nanotube biocorona and a functionalization method to prevent protein biofouling

Background: The intrinsic physicochemical properties of carbon nanotubes (CNTs) make them unique tools in nanotechnology. Their elemental composition, resilience, thermal properties, and surface reactivity make CNTs also of undisputed interest in biotechnology. In particular, their extraordinary ability to capture biomolecules on their surface makes them essential in this field. The proteins adsorbed on the CNTs create a biological coating that endows them the ability to interact with some cell receptors, penetrate membranes or interfere with cell biomechanics, thus behaving as an active bio-camouflage. But some of these proteins unfold, triggering an immune response that unpredictably changes the biological activity of CNTs. For this reason, the control of the biocorona is fundamental in the nanobiotechnology of CNTs. Results: Using TEM and AFM here we demonstrate a significant increase in CNTs diameter after protein functionalization. A quantitative analysis using TGA revealed that between 20 and 60% of the mass of functionalized nanotubes corresponds to protein, with single-walled CNTs capturing the highest amounts. To qualitatively/quantitatively characterize these biocoatings, we studied the biochemical "landscape" of the proteins captured by the different nanotubes after functionalization under various conditions. This study revealed a significant variability of the proteins in the corona as a function of the type of nanotube, the functionalization temperature, or the time after exposure to serum. Remarkably, the functionalization of a single type of CNT with sera from various human donors also resulted in different protein landscapes. Given the unpredictable assortment of proteins captured by the corona and the biological implications of this biocoating, we finally designed a method to genetically engineer and produce proteins to functionalize nanotubes in a controlled and customizable way. Conclusions: We demonstrate the high unpredictability of the spontaneous protein corona on CNTs and propose a versatile functionalization technique that prevents the binding of nonspecific proteins to the nanotube to improve the use of CNTs in biomedical applications.</p

Free-labeled nanoclay intracellular uptake tracking by confocal Raman imaging

Laponite is a nanoplatform that has been successfully used as a new biomaterial for drug delivery, tissue engineering and bioimaging at the nanoscale. In general, a deep knowledge of the mechanism interaction of the nanomaterial with biological components in a physiological environment is highly desirable for properly characterizing its therapeutic efficacy and toxicology. Up to know, the use of fluorescent dyes labelling both, the nanomaterial and cell components, has been a requirement to characterize the cell uptake and to visualize the entrance of the nanomaterial into the cytosol and the cell nucleus. The used of fluorophores usually perturb the physiological medium and can interfere in the nanomaterial cell interaction. A new Raman imaging methodology to track the uptake and internalization of Laponite nanoparticles into J774 macrophages line cells is presented in this work. The combination of Raman spectroscopy and confocal microscopy provides direct information about the localization of the nanoparticle into the cell, through its unique vibrational fingerprint without labelling or adding dyes, and taking advantage of the fact that Laponite and biological molecules bands can be clearly differentiated.We would like to thank IDIVAL for financial support, Projects N°NVAL16/17, INNVAL19/18 and NVAL18/07. CRL thanks the MINECO for the Juan de la Cierva Formación grant (ref. FJCI-2015-25306). This work has been supported by the Spanish MINECO, Instituto de Salud Carlos III, the European Union FEDER funds under Projects ref. PI16/00496 (AES 2016), PI19/00349 and DTS19/00033 (AES 2019). The authors are grateful to Dr F Madrazo and the Laser Microscopy Unit of the IDIVAL Institute for the use of the Confocal Raman Imaging Microscope