Cytotoxic Effects of Bilberry Extract on MCF7-GFP-Tubulin Breast Cancer Cells

Vy Nguyen,Jessica Tang,Emin Oroudjev,Choong Jae Lee,Cecilia Marasigan,Leslie Wilson,George Ayoub 한국식품영양과학회 2010 Journal of medicinal food Vol.13 No.2

Bilberry (European blueberry) has been reported to have many biological effects, including anticancer activity. In this study, we investigated the antiproliferative effects of bilberry extract in relation to its ability to induce apoptosis and affect microtubule assembly and organization in MCF7 human breast cancer cells. We observed that bilberry extract inhibited cell proliferation in a concentration-dependent fashion with a 50% inhibitory concentration of 0.3–0.4mg/mL, in concert with induction of apoptotic cell death. At these concentrations there was no selective inhibition of mitosis or any other cell cycle stage, nor was there any apparent effect on the microtubule or actin cytoskeletons. However, somewhat higher extract concentrations (0.5–0.9mg/mL) did cause an increase in the fraction of cells at the G2/M phase of the cell cycle, together with destruction of microtubules and formation of punctate tubulin aggregates in the cells. Bilberry extract at 0.3–0.4mg/mL did not appreciably inhibit microtubule polymerization in vitro, but significant inhibition of polymerization (30%) did occur at higher extract concentrations (0.5–1mg/mL). We conclude that bilberry extract as ingested by humans, not just the purified anthocyanins it contains, inhibits proliferation of and induces apoptosis in breast cancer cells at its lowest effective concentrations via a mechanism that does not involve action on microtubules or on mitosis. We further conclude that at somewhat higher concentrations the extract modifies microtubule organization in cells and causes accumulation of cells at mitosis by a direct action on microtubules.

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>

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>

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>

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>