(623aa) Determination of Progerin Lamin A Phase Association and Orientation to Phospholipid Membranes

Mutations in lamin A cause several disease states, called laminopathies. One of these, Hutchinson Gilford Progeria Syndrome (HGPS), produces a lamin A variant, progerin, that is improperly processed and accumulates at the nuclear membrane, triggering changes in gene expression, heterochromatin organization, as well as mechanical and morphological properties of the nucleus. Accumulation results in the formation of fibrous microdomains that alter nuclear mechanical behavior and overall cellular function. The molecular details of how the association of progerin lamin A with the nuclear membrane leads to the observed disease pathology is not known.

Membrane affinity of lamin A is regulated by a series of post-translational modifications that attach hydrophobic farnesyl and carboxy-methyl groups to the C-terminal cysteine group. Previous work has established that farnesylation alone is not sufficient for membrane association and implies other interactions contribute to the enhanced association of progerin lamin A. Enhanced membrane association is thought to be contributed by a terminal carboxy-methylation and polybasic stretch of histadines brought closer to the membrane surface by a 50 amino acid deletion (delta 50) that occurs on progerin lamin. This causes an accumulation of the delta 50 lamin A protein at the nuclear membrane in comparison to normal wild-type lamin A, as demonstrated in cells from HGPS patients with fluorescence and electron microscopy. We believe increased membrane retention enhances the aggregation of the lamin proteins into microdomains by preferential protein conformation and orientation at the membrane to maximize binding and simultaneously alters local membrane structure affecting downstream cellular signaling. We aim to determine the changes in protein conformation and orientation relative to the membrane surface caused by each membrane active moiety and change in membrane structure due to enhanced protein retention. To study lipid phase association of progerin lamin A we will use Giant Plasma Membrane Vesicles (GPMVs) Giant Unilammelar Vesicles (GUVs) and Langmuir Monolayers (LM) coupled with fluorophore labelled protein variants. To study protein orientation at the membrane interface we will use a combination of tethered bilayer membranes (tBLMs) and Langmuir monolayers.