Paclitaxel suppresses Tau-mediated microtubule bundling in a concentration-dependent manner

Choi, Myung Chul,Chung, Peter J.,Song, Chaeyeon,Miller, Herbert P.,Kiris, E.,Li, Youli,Wilson, Leslie,Feinstein, Stuart C.,Safinya, Cyrus R. Elsevier 2017 Biochimica et biophysica acta, General subjects Vol.1861 No.1

<P><B>Abstract</B></P> <P><B>Background</B></P> <P>Microtubules (MTs) are protein nanotubes comprised of straight protofilaments (PFs), head to tail assemblies of αβ-tubulin heterodimers. Previously, it was shown that Tau, a microtubule-associated protein (MAP) localized to neuronal axons, regulates the average number of PFs in microtubules with increasing inner radius <<I> R</I> <SUB>in</SUB> <SUP>MT</SUP> > observed for increasing Tau/tubulin-dimer molar ratio Φ<SUB>Tau</SUB> at paclitaxel/tubulin-dimer molar ratio Λ<SUB>Ptxl</SUB> =1/1.</P> <P><B>Methods</B></P> <P>We report a synchrotron SAXS and TEM study of the phase behavior of microtubules as a function of varying concentrations of paclitaxel (1/32≤Λ<SUB>Ptxl</SUB> ≤1/4) and Tau (human isoform 3RS, 0≤Φ<SUB>3RS</SUB> ≤1/2) at room temperature.</P> <P><B>Results</B></P> <P>Tau and paclitaxel have opposing regulatory effects on microtubule bundling architectures and microtubule diameter. Surprisingly and in contrast to previous results at Λ<SUB>Ptxl</SUB> =1/1 where microtubule bundles are absent, in the lower paclitaxel concentration regime (Λ<SUB>Ptxl</SUB> ≤1/4), we observe both microtubule doublets and triplets with increasing Tau. Furthermore, increasing paclitaxel concentration (up to Λ<SUB>Ptxl</SUB> =1/1) slightly decreased the average microtubule diameter (by ~1 PF) while increasing Tau concentration (up to Φ<SUB>3RS</SUB> =1/2) significantly increased the diameter (by ~2–3 PFs).</P> <P><B>Conclusions</B></P> <P>The suppression of Tau-mediated microtubule bundling with increasing paclitaxel is consistent with paclitaxel seeding more, but shorter, microtubules by rapidly exhausting tubulin available for polymerization. Microtubule bundles require the aggregate Tau-Tau attractions along the microtubule length to overcome individual microtubule thermal energies disrupting bundles.</P> <P><B>General significance</B></P> <P>Investigating MAP-mediated interactions between microtubules (as it relates to <I>in vivo</I> behavior) requires the elimination or minimization of paclitaxel.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Increasing paclitaxel suppresses Tau-mediated microtubule bundling. </LI> <LI> A length-dependent mechanism for Tau-mediated microtubule bundling is proposed. </LI> <LI> Understanding MAP/microtubule behavior requires elimination of paclitaxel use. </LI> </UL> </P>

Screening of key miRNAs related with the differentiation of subcutaneous adipocytes and the validation of miR-133a-3p functional significance in goats

Li Xin,Zhang Hao,Wang Yong,Li Yanyan,Wang Youli,Zhu Jiangjiang,Lin Yaqiu 아세아·태평양축산학회 2023 Animal Bioscience Vol.36 No.1

Objective: Adipocyte differentiation is regulated by a variety of functional genes and noncoding RNAs. However, the role of miRNAs in lipid deposition of goat white adipose tissue is still unclear. Therefore, this study revealed the miRNA expression profile in goat subcutaneous adipocytes by sRNA-seq. Methods: The miRNA expressed in goat subcutaneous preadipocytes and the mature adipocytes were sequenced by sRNA-seq. The differentially expressed miRNAs (DEm) were screened and gene ontology (GO) and Kyoto encyclopedia for genes and genomes (KEGG) analyses were performed. Gain-of-function and loss-of-function combined with oil red O staining, Bodipy staining, and quantitative reverse-transcription polymerase chain reaction (qPCR) were utilized to determine the effect of miR-133a-3p on adipocyte differentiation. Results: A total of 218 DEm were screened out. The target genes of these DEm were significantly enriched in GO items such as biological regulation and in KEGG terms such as FAK signaling pathway and MAPK signaling pathway. qPCR verified that the expression trend of miRNA was consistent with miRNA-seq. The gain-of-function or loss-of-function of miR-133a-3p showed that it promoted or inhibited the accumulation of lipid droplets, and CCAAT enhancer binding protein α (C/EBPα) and C/EBPβ were extremely significantly up-regulated or down-regulated respectively (p<0.01), the loss-of-function also led to a significant down-regulation of peroxisome proliferator activated receptor gamma (PPARγ) (p<0.01). Conclusion: This study successfully identified miRNAs expression patterns in goat subcutaneous adipocytes, and functional identification indicates that miR-133a-3p is a positive regulator of the differentiation process of goat subcutaneous adipocytes. Our results lay the foundation for the molecular mechanism of lipid deposition in meat-source goats from the perspective of miRNA.

Response of Manas River fluvial landforms to tectonic movement, at the north flank of the Tianshan Mountain, China

Xingmin Shi,Youli Li,Jingchun Yang 한국지질과학협의회 2010 Geosciences Journal Vol.14 No.3

Manas River is the largest river at the north foot of Tianshan Mountain, and it is obvious that the landforms at the north flank of Tianshan Mountains respond to the tectonic movement. A hypothesis has been put forward that geomorphology of the north flank of the Tianshan Mountain Range is the result of active tectonic processes. Based on the filed survey and analysis, the hypothesis has been proved by the results of the research. By investigating into the evolutions of Manas River valley fold hills, alluvial fans and terraces, it is indicated that Tianshan Mountain piedmont has been constantly uplifted along with main body of Tianshan Mountain since Quaternary, and continues to be thrust from south to north, which makes the piedmont range constantly extend to the basin direction. The deformed main body also moves from south to north. Meanwhile, the alternant Pleistocene ice age and interglacial age climate also has significant impact on the current landform patterns of north piedmont of Tianshan Mountain.

1LD-7 The effect of multivalent cations on paclitaxel-stabilized microtubule assembly, disassembly, and structure

송채연,( Cyrus R. Safinya ),( Peter J. Chung ),( Youli Li ),( Kai K. Ewert ),( Myung Chul Choi ) 한국공업화학회 2017 한국공업화학회 연구논문 초록집 Vol.2017 No.1

We focus on the effects of multivalent cations, and separately, of microtubule-associated protein (MAP) Tau, on microtubule (MT) ordering (bundling), MT disassembly, and MT structure. Counter-ion directed bundling of paclitaxel-stabilized MTs is a model electrostatic system, which parallels efforts to understand MT bundling by intrinsically disordered proteins expressed in neurons. We describe studies, which reveal an unexpected transition from tightly spaced MT bundles to loose bundles consisting of strings of MTs as the valence of the cationic counter-ion decreases from Z=3 to Z=2. In a more biologically related system we review synchrotron small angle x-ray scattering (SAXS) studies on the effect of the Tau on the structure of paclitaxel-stabilized MTs.

Structural Evolution of Environmentally Responsive Cationic Liposome–DNA Complexes with a Reducible Lipid Linker

Shirazi, Rahau S.,Ewert, Kai K.,Silva, Bruno F. B.,Leal, Cecilia,Li, Youli,Safinya, Cyrus R. American Chemical Society 2012 Langmuir Vol.28 No.28

<P>Environmentally responsive materials (i.e., materials that respond to changes in their environment with a change in their properties or structure) are attracting increasing amounts of interest. We recently designed and synthesized a series of cleavable multivalent lipids (CMVLn, with <I>n</I> = 2–5 being the number of positive headgroup charges at full protonation) with a disulfide bond in the linker between their cationic headgroup and hydrophobic tails. The self-assembled complexes of the CMVLs and DNA are a prototypical environmentally responsive material, undergoing extensive structural rearrangement when exposed to reducing agents. We investigated the structural evolution of CMVL–DNA complexes at varied complex composition, temperature, and incubation time using small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS). A related lipid with a stable linker, TMVL4, was used as a control. In a nonreducing environment, CMVL–DNA complexes form the lamellar (L<SUB>α</SUB><SUP>C</SUP>) phase, with DNA rods sandwiched between lipid bilayers. However, new self-assembled phases form when the disulfide linker is cleaved by dithiothreitol or the biologically relevant reducing agent glutathione. The released DNA and cleaved CMVL headgroups form a loosely organized phase, giving rise to a characteristic broad SAXS correlation profile. CMVLs with high headgroup charge also form condensed DNA bundles. Intriguingly, the cleaved hydrophobic tails of the CMVLs reassemble into tilted chain-ordered L<SUB>β′</SUB> phases upon incubation at physiological temperature (37 °C), as indicated by characteristic WAXS peaks. X-ray scattering further reveals that two of the three phases (L<SUB>βF</SUB>, L<SUB>βL</SUB>, and L<SUB>βI</SUB>) constituting the L<SUB>β′</SUB> phase coexist in these samples. The described system may have applications in lipid-based nanotechnologies.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/langd5/2012/langd5.2012.28.issue-28/la301181b/production/images/medium/la-2012-01181b_0001.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/la301181b'>ACS Electronic Supporting Info</A></P>

Ion specific effects in bundling and depolymerization of taxol-stabilized microtubules

Needleman, Daniel J.,Ojeda-Lopez, Miguel A.,Raviv, Uri,Miller, Herbert P.,Li, Youli,Song, Chaeyeon,Feinstein, Stuart C.,Wilson, Leslie,Choi, Myung Chul,Safinya, Cyrus R. The Royal Society of Chemistry 2013 Faraday discussions Vol.166 No.-

<P>Microtubules (MTs) are nanometer scale hollow cylindrical biological polyelectrolytes. They are assembled from α/β-tubulin dimers, which stack to form protofilaments (PFs) with lateral interactions between PFs resulting in the curved MT. In cells, MTs and their assemblies are critical components in a range of functions from providing tracks for the transport of cargo to forming the spindle structure during mitosis. Previous studies have shown that while cations with valence equal to or larger than 3+ tend to assemble tight 3D bundles of taxol-stabilized MTs, certain divalent cations induce relatively loose 2D bundles of different symmetry (D. J. Needleman <I>et al.</I>, <I>Proc. Natl. Acad. Sci. U. S. A.</I>, 2004, <B>101</B>, 16099). Similarly, divalent cations form 2D bundles of DNA adsorbed on cationic membranes (I. Koltover <I>et al.</I>, <I>Proc. Natl. Acad. Sci. U. S. A.</I>, 2000, <B>97</B>, 14046). The bundling behavior for these biological polyelectrolyte systems is qualitatively in agreement with current theory. Here, we present results which show that, unlike the case for DNA adsorbed on cationic membranes, bundling of taxol-stabilized MTs occurs only for certain divalent cations above a critical ion concentration (<I>e.g.</I> Ca<SUP>2+</SUP>, Sr<SUP>2+</SUP>, Ba<SUP>2+</SUP>). Instead, many divalent cations pre-empt the bundling transition and depolymerize taxol-stabilized MTs at a lower counterion concentration. Although previous cryogenic TEM has shown that, in the absence of taxol, Ca<SUP>2+</SUP> depolymerizes MTs assembling in buffers containing GTP (guanosine triphosphate), our finding is surprising given the known stabilizing effects of taxol on GDP (guanosine diphosphate)-MTs. The ion concentration required for MT depolymerization decreases with increasing atomic number for the divalents Mg<SUP>2+</SUP>, Mn<SUP>2+</SUP>, Co<SUP>2+</SUP>, and Zn<SUP>2+</SUP>. GdCl<SUB>3</SUB> (3+) is found to be extremely efficient at MT depolymerization requiring ion concentrations of about 1 mM, while oligolysine (2+), is observed not to depolymerize MTs at concentrations as high as 144 mM. The surprising MT depolymerization results are discussed in the context of divalents either disrupting lateral interactions between PFs (which are strengthened for taxol containing β-tubulin), or interfering with taxol's ability to induce flexibility at the interface between two tubulin dimers in the same PF (which has been recently suggested as a mechanism by which taxol stabilizes MTs post-hydrolysis with the induced flexibility counteracting the kink between GDP-tubulin dimers in a PF).</P>

Minireview - Microtubules and Tubulin Oligomers: Shape Transitions and Assembly by Intrinsically Disordered Protein Tau and Cationic Biomolecules

Safinya, Cyrus R.,Chung, Peter J.,Song, Chaeyeon,Li, Youli,Miller, Herbert P.,Choi, Myung Chul,Raviv, Uri,Ewert, Kai K.,Wilson, Leslie,Feinstein, Stuart C. American Chemical Society 2019 Langmuir Vol.35 No.48

<P>In this minireview, which is part of a special issue in honor of Jacob N. Israelachvili’s remarkable research career on intermolecular forces and interfacial science, we present studies of structures, phase behavior, and forces in reaction mixtures of microtubules (MTs) and tubulin oligomers with either intrinsically disordered protein (IDP) Tau, cationic vesicles, or the polyamine spermine (4+). Bare MTs consist of 13 protofilaments (PFs), on average, where each PF is made of a linear stack of αβ-tubulin dimers (i.e., tubulin oligomers). We begin with a series of experiments which demonstrate the flexibility of PFs toward shape changes in response to local environmental cues. First, studies show that MT-associated protein (MAP) Tau controls the diameter of microtubules upon binding to the outer surface, implying a shape change in the cross-sectional area of PFs forming the MT perimeter. The diameter of a MT may also be controlled by the charge density of a lipid bilayer membrane that coats the outer surface. We further describe an experimental study where it is unexpectedly found that the biologically relevant polyamine spermine (+4e) is able to depolymerize <I>taxol-stabilized</I> microtubules with efficiency that increases with decreasing temperature. This MT destabilization drives a dynamical structural transition where inside-out curving of PFs, during the depolymerization peeling process, is followed by reassembly of ring-like curved PF building blocks into an array of helical inverted tubulin tubules. We finally turn to a very recent study on pressure-distance measurements in bundles of MTs employing the small-angle X-ray scattering (SAXS)-osmotic pressure technique, which complements the surface-forces-apparatus technique developed by Jacob N. Israelachvili. These latter studies are among the very few which are beginning to shed light on the precise nature of the interactions between MTs mediated by MAP Tau in 37 °C reaction mixtures containing GTP and lacking taxol.</P> [FIG OMISSION]</BR>

Two-Dimensional Packing of Short DNA with Nonpairing Overhangs in Cationic Liposome–DNA Complexes: From Onsager Nematics to Columnar Nematics with Finite-Length Columns

Bouxsein, Nathan F.,Leal, Cecí,lia,McAllister, Christopher S.,Ewert, Kai K.,Li, Youli,Samuel, Charles E.,Safinya, Cyrus R. American Chemical Society 2011 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.133 No.19

<P>We report the formation of liquid crystalline (LC) phases of short double-stranded DNA with nonpairing (nonsticky) overhangs, confined between two-dimensional (2D) lipid bilayers of cationic liposome–DNA complexes. In a landmark study (<I>Science</I><B>2007</B>, <I>318</I>, 1276), Nakata et al. reported on the discovery of strong end-to-end stacking interactions between short DNAs (sDNAs) with blunt ends, leading to the formation of 3D nematic (N) and columnar LC phases. Employing synchrotron small-angle X-ray scattering, we have studied the interplay between shape anisotropy-induced and DNA end-to-end interaction-induced N ordering for 11, 24, and 48 bp sDNA rods with single-stranded oligo-thymine (T) overhangs modulating the end-to-end interactions. For suppressed stacking interactions with 10-T overhangs, the volume fraction of sDNA at which the 2D isotropic (I)-to-N transition occurs for 24 and 48 bp sDNA rods depended on their length-to-width (<I>L</I>/<I>D</I>) shape anisotropy, qualitatively consistent with Onsager’s theory for the entropic alignment of rigid rods. As the overhang length is reduced from 10 to 5 and 2 T for 24 and 48 bp sDNA, the N-to-I transition occurs at lower volume fractions, indicating the onset of some degree of end-to-end stacking interactions. The 11 bp sDNA rods with 5- and 10-T overhangs remain in the I phase, consistent with their small shape anisotropy (<I>L</I>/<I>D</I> ≈ 1.9) below the limit for Onsager LC ordering. Unexpectedly, in contrast to the behavior of 24 and 48 bp sDNA, the end-to-end interactions between 11 bp sDNA rods with 2-T overhangs set in dramatically, and a novel 2D columnar N phase (N<SUB>C</SUB>) with finite-length columns formed. The building blocks of this phase are comprised of 1D stacks of (on average) four 11 bp DNA-2T rods with an effective <I>L</I><SUB>stacked</SUB>/<I>D</I> ≈ 8.2. Our findings have implications for the DNA-directed assembly of nanoparticles on 2D platforms via end-to-end interactions and in designing optimally packed LC phases of short anisotropic biomolecules (such as peptides and short-interfering RNAs) on nanoparticle membranes, which are used in gene silencing and chemical delivery.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/2011/jacsat.2011.133.issue-19/ja202082c/production/images/medium/ja-2011-02082c_0010.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ja202082c'>ACS Electronic Supporting Info</A></P>

Direct force measurements reveal that protein Tau confers short-range attractions and isoform-dependent steric stabilization to microtubules

Chung, Peter J.,Choi, Myung Chul,Miller, Herbert P.,Feinstein, H. Eric,Raviv, Uri,Li, Youli,Wilson, Leslie,Feinstein, Stuart C.,Safinya, Cyrus R. National Academy of Sciences 2015 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.112 No.47

<P><B>Significance</B></P><P>The microtubule-associated protein Tau is known to stabilize microtubules against depolymerization in neuronal axons, ensuring proper trafficking of organelles along microtubules in long axons. Abnormal interactions between Tau and microtubules are implicated in Alzheimer’s disease and other neurodegenerative disorders. We directly measured forces between microtubules coated with Tau isoforms by synchrotron small-angle X-ray scattering of reconstituted Tau–microtubule mixtures under osmotic pressure (mimicking molecular crowding in cells). We found that select Tau isoforms fundamentally alter forces between microtubules by undergoing a conformational change on microtubule surfaces at a coverage indicative of an unusually extended Tau state. This gain of function by longer isoforms in imparting steric stabilization to microtubules is essential in preventing microtubule aggregation and loss of function in organelle trafficking.</P><P>Microtubules (MTs) are hollow cytoskeletal filaments assembled from αβ-tubulin heterodimers. Tau, an unstructured protein found in neuronal axons, binds to MTs and regulates their dynamics. Aberrant Tau behavior is associated with neurodegenerative dementias, including Alzheimer’s. Here, we report on a direct force measurement between paclitaxel-stabilized MTs coated with distinct Tau isoforms by synchrotron small-angle X-ray scattering (SAXS) of MT-Tau mixtures under osmotic pressure (<I>P</I>). In going from bare MTs to MTs with Tau coverage near the physiological submonolayer regime (Tau/tubulin-dimer molar ratio; Φ<SUB>Tau</SUB> = 1/10), isoforms with longer N-terminal tails (NTTs) sterically stabilized MTs, preventing bundling up to <I>P</I><SUB>B</SUB> ∼ 10,000–20,000 Pa, an order of magnitude larger than bare MTs. Tau with short NTTs showed little additional effect in suppressing the bundling pressure (<I>P</I><SUB>B</SUB> ∼ 1,000–2,000 Pa) over the same range. Remarkably, the abrupt increase in <I>P</I><SUB>B</SUB> observed for longer isoforms suggests a mushroom to brush transition occurring at 1/13 < Φ<SUB>Tau</SUB> < 1/10, which corresponds to MT-bound Tau with NTTs that are considerably more extended than SAXS data for Tau in solution indicate. Modeling of Tau-mediated MT–MT interactions supports the hypothesis that longer NTTs transition to a polyelectrolyte brush at higher coverages. Higher pressures resulted in isoform-independent irreversible bundling because the polyampholytic nature of Tau leads to short-range attractions. These findings suggest an isoform-dependent biological role for regulation by Tau, with longer isoforms conferring MT steric stabilization against aggregation either with other biomacromolecules or into tight bundles, preventing loss of function in the crowded axon environment.</P>

Nanoscale Assembly in Biological Systems: From Neuronal Cytoskeletal Proteins to Curvature Stabilizing Lipids

Safinya, Cyrus R.,Raviv, Uri,Needleman, Daniel J.,Zidovska, Alexandra,Choi, Myung Chul,Ojeda‐,Lopez, Miguel A.,Ewert, Kai K.,Li, Youli,Miller, Herbert P.,Quispe, Joel,Carragher, Bridget,Potter, WILEY‐VCH Verlag 2011 ADVANCED MATERIALS Vol.23 No.20

<P><B>Abstract</B></P><P>The review will describe experiments inspired by the rich variety of bundles and networks of interacting microtubules (MT), neurofilaments, and filamentous‐actin in neurons where the nature of the interactions, structures, and structure‐function correlations remain poorly understood. We describe how three‐dimensional (3D) MT bundles and 2D MT bundles may assemble, in cell free systems in the presence of counter‐ions, revealing structures not predicted by polyelectrolyte theories. Interestingly, experiments reveal that the neuronal protein tau, an abundant MT‐associated‐protein in axons, modulates the MT diameter providing insight for the control of geometric parameters in bio‐ nanotechnology. In another set of experiments we describe lipid‐protein‐nanotubes, and lipid nano‐ tubes and rods, resulting from membrane shape evolution processes involving protein templates and curvature stabilizing lipids. Similar membrane shape changes, occurring in cells for the purpose of specific functions, are induced by interactions between membranes and proteins. The biological materials systems described have applications in bio‐nanotechnology.</P>