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>

Human Microtubule-Associated-Protein Tau Regulates the Number of Protofilaments in Microtubules: A Synchrotron X-Ray Scattering Study

Choi, M.C.,Raviv, U.,Miller, H.P.,Gaylord, M.R.,Kiris, E.,Ventimiglia, D.,Needleman, D.J.,Kim, M.W.,Wilson, L.,Feinstein, S.C.,Safinya, C.R. Biophysical Society ; Published for the Biophysica 2009 Biophysical journal Vol.97 No.2

Microtubules (MTs), a major component of the eukaryotic cytoskeleton, are 25 nm protein nanotubes with walls comprised of assembled protofilaments built from αβ heterodimeric tubulin. In neural cells, different isoforms of the microtubule-associated-protein (MAP) tau regulate tubulin assembly and MT stability. Using synchrotron small angle x-ray scattering (SAXS), we have examined the effects of all six naturally occurring central nervous system tau isoforms on the assembly structure of taxol-stabilized MTs. Most notably, we found that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius <R<SUP>MT</SUP>> of MTs with increasing Φ, the tau/tubulin-dimer molar ratio. Within experimental scatter, the change in <R<SUP>MT</SUP>> seems to be isoform independent. Significantly, <R<SUP>MT</SUP>> was observed to rapidly increase for 0 < Φ < 0.2 and saturate for Φ between 0.2-0.5. Thus, a local shape distortion of the tubulin dimer on tau binding, at coverages much less than a monolayer, is spread collectively over many dimers on the scale of protofilaments. This implies that tau regulates the shape of protofilaments and thus the spontaneous curvature C<SUB>o</SUB><SUP>MT</SUP> of MTs leading to changes in the curvature C<SUP>MT</SUP> (=1/R<SUP>MT</SUP>). An important biological implication of these findings is a possible allosteric role for tau where the tau-induced shape changes of the MT surface may effect the MT binding activity of other MAPs present in neurons. Furthermore, the results, which provide insight into the regulation of the elastic properties of MTs by tau, may also impact biomaterials applications requiring radial size-controlled nanotubes.

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>

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>