Jeffrey F. Herbstman

Luminal Localization of α-tubulin K40 Acetylation by Cryo-EM Analysis of Fab-Labeled Microtubules

The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs) that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40) has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.

Nanochannels fabricated by high-intensity femtosecond laser pulses on dielectric surfaces

Direct scanning electron microscopy examination reveals a complex structure of narrow, micron-deep, internal nanochannels within shallow, nanoscale, external craters fabricated on glass and sapphire surfaces by single high-intensity femtosecond laser pulses, with nearly the same intensity thresholds for both features. Formation of the channels is accompanied by extensive expulsion of molten material produced via surface spallation and phase explosion mechanisms, and redeposited around the corresponding external craters. Potential mechanisms underlying fabrication of the unexpectedly deep channels in dielectrics are considered.

Paclitaxel-Conjugated PAMAM Dendrimers Adversely Affect Microtubule Structure through Two Independent Modes of Action

Paclitaxel (Taxol) is an anticancer drug that induces mitotic arrest via microtubule hyperstabilization but causes side effects due to its hydrophobicity and cellular promiscuity. The targeted cytotoxicity of hydrophilic paclitaxel-conjugated polyamidoamine (PAMAM) dendrimers has been demonstrated in cultured cancer cells. Mechanisms of action responsible for this cytotoxicity are unknown, that is, whether the cytotoxicity is due to paclitaxel stabilization of microtubules, as is whether paclitaxel is released intracellularly from the dendrimer. To determine whether the conjugated paclitaxel can bind microtubules, we used a combination of ensemble and single microtubule imaging techniques in vitro. We demonstrate that these conjugates adversely affect microtubules by (1) promoting the polymerization and stabilization of microtubules in a paclitaxel-dependent manner, and (2) bundling preformed microtubules in a paclitaxel-independent manner, potentially due to protonation of tertiary amines in the dendrimer interior. Our results provide mechanistic insights into the cytotoxicity of paclitaxel-conjugated PAMAM dendrimers and uncover unexpected risks of using such conjugates therapeutically.

High-aspect ratio nanochannel formation by single femtosecond laser pulses

Single femtosecond pulsed laser damage can be confined radially to regions smaller than the focus spot size due to the highly nonlinear mechanisms for energy absorption and ablation in transparent dielectrics. Along the propagation axis, however, we show that channels can be machined much deeper than the Rayleigh range of the laser focus. Using focused ion beam cross sections and acetate imprints, we analyze these channels and show that spherical aberration is not the primary source for this elongated damage, which is likely caused by microscale filamentation.

ERdj5 Reductase Cooperates with Protein Disulfide Isomerase To Promote Simian Virus 40 Endoplasmic Reticulum Membrane Translocation

ABSTRACT The nonenveloped polyomavirus (PyV) simian virus 40 (SV40) traffics from the cell surface to the endoplasmic reticulum (ER), where it penetrates the ER membrane to reach the cytosol before mobilizing into the nucleus to cause infection. Prior to ER membrane penetration, ER lumenal factors impart structural rearrangements to the virus, generating a translocation-competent virion capable of crossing the ER membrane. Here we identify ERdj5 as an ER enzyme that reduces SV40s disulfide bonds, a reaction important for its ER membrane transport and infection. ERdj5 also mediates human BK PyV infection. This enzyme cooperates with protein disulfide isomerase (PDI), a redox chaperone previously implicated in the unfolding of SV40, to fully stimulate membrane penetration. Negative-stain electron microscopy of ER-localized SV40 suggests that ERdj5 and PDI impart structural rearrangements to the virus. These conformational changes enable SV40 to engage BAP31, an ER membrane protein essential for supporting membrane penetration of the virus. Uncoupling of SV40 from BAP31 traps the virus in ER subdomains called foci, which likely serve as depots from where SV40 gains access to the cytosol. Our study thus pinpoints two ER lumenal factors that coordinately prime SV40 for ER membrane translocation and establishes a functional connection between lumenal and membrane events driving this process. IMPORTANCE PyVs are established etiologic agents of many debilitating human diseases, especially in immunocompromised individuals. To infect cells at the cellular level, this virus family must penetrate the host ER membrane to reach the cytosol, a critical entry step. In this report, we identify two ER lumenal factors that prepare the virus for ER membrane translocation and connect these lumenal events with events on the ER membrane. Pinpointing cellular components necessary for supporting PyV infection should lead to rational therapeutic strategies for preventing and treating PyV-related diseases.

Morphologies and nonlinear scaling of laser damage on glass surfaces by tightly focused femtosecond pulses

We examine the relationship between pulse energy and the morphology of damage by a femtosecond pulsed laser, tightly focused onto the back surface of glass. For fluences up to three times that of threshold, an unexpected discontinuity in the scaling of damage size is caused by ejection of rings of material surrounding central damage that appear above a sharp threshold fluence. A mechanism for the production of these structures via thermal expansion and shockwave generation is proposed.

Femtosecond nanomachining: theory and applications in biomedical research and analysis

The nonlinear mechanisms of femtosecond laser damage allow tight control of ablation to precisely remove very small amounts of material, leaving holes as small as tens of nanometers wide. By serially targeting laser pulses in glass, a host of three dimensional nano- and microfluidic structures can be formed including nozzles, mixers, and separation columns. Recent advances allow the formation of high aspect ratio nanochannels from single pulses, thus helping address fabrication speed limitations presented by serial processing. Femtosecond nanomachining is enabling for a variety of applications including nanoscale devices for analytic separations, chemical analysis, and biomedical diagnostics.

Taxol-Conjugated Pamam Dendrimers Utilize Three Modes of Action on Microtubule Structure

Paclitaxel (Taxol) is a cancer drug that causes cell death by stabilizing microtubules and consequently arresting cell division. Previously, the cytotoxicity of taxol-conjugated PAMAM dendrimers was demonstrated in cancer cells. The exact mode(s) of action responsible for the potent cytotoxicity of these dendrimers were not examined but the literature provides uncertainty that the taxol could be released from the dendrimer carrier after cellular entry. Accordingly, we asked whether the taxol-dendrimer conjugate itself is able to bind microtubules. To address this question, we investigated the effect of these conjugates on microtubules in vitro using total internal reflection fluorescence microscopy (TIRFM) and transmission electron microscopy (TEM). We find that the taxol-dendrimer conjugate affects microtubule structure in two ways: (1) the conjugate can bind tubulin during tubulin polymerization and stabilize it into a tubular structure and (2) the conjugate can bundle microtubules in a manner that is not dependent on taxol, but dendrimer electrostatics. Both of these modes of action would arrest cell division and consequently kill the cell. This is the first time that the binding of taxol-conjugated dendrimers to microtubules has been demonstrated in vitro. Furthermore, our results provide both mechanistic insights into the cytotoxicity of the previously characterized taxol-conjugated PAMAM dendrimers and additional evidence for the potential of these and similar conjugates as cancer therapeutics.

Single femtosecond pulse nanochannel formation in glass

Single pulse femtosecond laser damage in transparent dielectrics has been shown to occur through nonlinear damage mechanisms that can allow material removal on scales well below the classical limit of the order of the wavelength of the incident light. These mechanisms can be harnessed to allow the optical machining of devices on the nanoscale. We observe the formation of high aspect-ratio nanochannels by single femtosecond pulses. These channels, several microns in length, can be formed at the front or rear surface of a sample, corresponding to conditions under which spherical aberration is expected and where it is minimized. The presence of similar channels at both locations suggests that aberration does not play a critical role in nanochannel creation, and we present evidence supporting a dominant role of self focusing and microscale filamentation. Applications for these long nanoscale diameter channels include nanopores, nanowells, or out-of-plane vias. The ability to generate these channels with single pulses allows rapid fabrication that complements existing techniques, thus addressing a major limitation to fabrication of microfluidics and nanopores.

Ejection of glass rings during tightly focused femtosecond laser damage at a glass surface

We observe a discontinuity in the scaling between the size of damage and the pulse energy for femtosecond laser pulses tightly focused at a glass surface. This discontinuity corresponds to the threshold for formation and ejection of rings of material surrounding the focus center. The mechanism for the generation of these structures appears distinct from that of the central holes and is ascribed to subsurface absorption leading to thermal expansion and shock wave formation.