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Functionalizing carbon nanotubes as a model to study the actin-based mechanism used by baculovirus to enter the cell nucleus

University of British Columbia

The cell nucleus is protected by an impermeable nuclear envelope (NE) containing nuclear pore complexes (NPCs), through which controlled trafficking of molecules between the cytoplasm and the nucleus occurs. Baculovirus nucleocapsids, which are rod-shaped DNA containing capsids, have the ability to cross the NPC to deliver their genome into the nucleus. However, baculovirus nucleocapsids do not have signaling motifs required for conventional signal mediated nuclear import. Furthermore, they are considered to be too large for passive diffusion through the NPC. Recently, our laboratory has proposed that baculovirus nucleocapsids use an unconventional strategy to enter the nucleus via actin propulsion. A Wiskott-Aldrich syndrome protein (WASP)-like protein on one end of the baculovirus nucleocapsid hijacks the actin polymerization machinery of the host cell to promote the formation of actin comet tails that propel the nucleocapsid through the NPC. The main aim of this thesis is to test whether multiwalled carbon nanotubes (MWCNTs), which like baculovirus nucleocapsids are rod-shaped, can enter the nucleus using actin-based propulsion. I have accomplished this by functionalizing MWCNTs, enabling them to induce actin polymerization, similar to baculovirus nucleocapsids. MWCNTs with baculovirus nucleocapsid-like diameters were conjugated to WASP. Successful protein conjugation to MWCNTs was confirmed using immuno-gold electron microscopy. Similar levels of MWCNT-WASP and our corresponding control complex, MWCNT-BSA-Cy3, were present inside HeLa cells; however, their ability to enter the nuclei was significantly different. Nuclear MWCNT-WASP was detected in more than half the cell population. In contrast, MWCNT-BSA-Cy3 was detected in only a few nuclei. Successful nuclear entry of MWCNT-WASP was also detected in semi-permeabilized cells. Moreover, disrupting actin polymerization noticeably decreased nuclear entry of MWCNT-WASP. Furthermore, isolated nuclei were incubated with MWCNT-WASP and G-actin under iii conditions of actin polymerization. Following this, confocal imaging depicted physical depressions of the NE at the sites where MWCNT-WASPs were docked. Taken together, these results indicate that mechanical force generated by actin-based propulsion can drive the nuclear entry of MWCNT-WASP. This supports the conclusion that the baculovirus nucleocapsid uses actin-based propulsion to enter the nucleus through the NPC.

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2019-03-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/64835

Zoology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/