Huntingtin associates with the actin cytoskeleton and α-actinin isoforms to influence stimulus dependent morphology changes

Abstract

One response of cells to growth factor stimulus involves changes in morphology driven by the actin cytoskeleton and actin associated proteins which regulate functions such as cell adhesion, motility and in neurons, synaptic plasticity. Previous studies suggest that Huntingtin may be involved in regulating morphology however, there has been limited evidence linking endogenous Huntingtin localization or function with cytoplasmic actin in cells. We found that depletion of Huntingtin in human fibroblasts reduced adhesion and altered morphology and these phenotypes were made worse with growth factor stimulation, whereas the presence of the Huntington’s Disease mutation inhibited growth factor induced changes in morphology and increased numbers of vinculin-positive focal adhesions. Huntingtin immunoreactivity localized to actin stress fibers, vinculin-positive adhesion contacts and membrane ruffles in fibroblasts. Interactome data from others has shown that Huntingtin can associate with α-actinin isoforms which bind actin filaments. Mapping studies using a cDNA encoding α-actinin-2 showed that it interacts within Huntingtin aa 399–969. Double-label immunofluorescence showed Huntingtin and α-actinin-1 co-localized to stress fibers, membrane ruffles and lamellar protrusions in fibroblasts. Proximity ligation assays confirmed a close molecular interaction between Huntingtin and α-actinin-1 in human fibroblasts and neurons. Huntingtin silencing with siRNA in fibroblasts blocked the recruitment of α-actinin-1 to membrane foci. These studies support the idea that Huntingtin is involved in regulating adhesion and actin dependent functions including those involving α-actinin.

Genistein induces degradation of mutant huntingtin in fibroblasts from Huntington’s disease patients

“…We observed that in HD fibroblasts, mHTT formed aggregates that associated with cytoskeletal fibers. Such results are compatible with recently reported observations that HTT can be associated with isoforms of α-actinin that bind actin filaments, and that mHTT localizes specifically to actin stress fibers (Tousley et al 2019 ). Again, treatment with genistein resulted in disappearance of aggregates, and HTT distribution resembling the distribution in healthy cells.…”

Disposition of Proteins and Lipids in Synaptic Membrane Compartments Is Altered in Q175/Q7 Huntington’s Disease Mouse Striatum

“…In addition, mutant Huntingtin may also indirectly affect synaptic function by changing transcription of synaptic genes ( Langfelder et al, 2016 ) or have pleiomorphic effects. For instance, alpha-actinin-2, which is an actin bundling protein, interacts with Huntingtin ( Tousley et al, 2019a ) and also interacts with NMDA 2b in spines ( Wyszynski et al, 1997 ). Alpha-actinin-2 is also transcriptionally downregulated in HD models ( Langfelder et al, 2016 ) which may create instability in spines and impact NMDA 2B receptors distribution.…”

“…It is possible that during preparation of the fractions in our study homogenization weakened structural integrity to give rise to the altered axon terminal and vesicle morphology we observed. Huntingtin has been implicated in actin morphology directly through interactions with actin ( Angeli et al, 2010 ) and interactions with actin binding proteins such as profilin, cofilin, alpha-actinins ( Culver et al, 2012 ; Shirasaki et al, 2012 ; Tourette et al, 2014 ; Tousley et al, 2019a ) and mutant Huntingtin can exert negative effects through ROCK ( Shao et al, 2008 ), Rac1 ( Tousley et al, 2019b ) and potentially other interacting proteins such as HIP1 and GIT1 ( Harjes and Wanker, 2003 ; Goehler et al, 2004 ). Alternatively, failure of vesicles to bud due to impaired Rab11 dependent trafficking in the presence of mutant Huntingtin ( Li et al, 2009 ) may account for the change in distribution in size of measured particles.…”

Huntington’s Disease—An Outlook on the Interplay of the HTT Protein, Microtubules and Actin Cytoskeletal Components

“…However, immunofluorescence studies have not confirmed those findings. The functional interaction of HTTs with α-actinin isoforms has confirmed by the integrated approach—immunoprecipitation method using exogenously expressed proteins in combination with immunofluorescence and PLA analysis [ 113 ]. The authors suggest that HTT regulates the localization of α-actinin-1 and combines growth factor signaling with actin polymerization and the formation of actin bundles in newly formed adhesion sites.…”