2 research outputs found
Semitransparent Nanostructured Films for Imaging Mass Spectrometry and Optical Microscopy
Semitransparent porous silicon substrates have been developed
for
pairing nanostructure-initiator mass spectrometry (NIMS) imaging with
traditional optical-based microscopy techniques. Substrates were optimized
to generate the largest NIMS signal while maintaining sufficient transparency
to allow visible light to pass through for optical microscopy. Using
these substrates, both phase-contrast and NIMS images of phospholipids
from a scratch-wounded cell monolayer were obtained. NIMS images were
generated using a spatial resolution of 14 μm. Coupled with
further improvements in spatial resolution, this approach may allow
for the localization of intact biological molecules within cells without
the need for labeling
Phosphorylation of Serine 106 in Asef2 Regulates Cell Migration and Adhesion Turnover
Asef2, a 652-amino acid protein,
is a guanine nucleotide exchange
factor (GEF) that regulates cell migration and other processes via
activation of Rho family GTPases, including Rac. Binding of the tumor
suppressor adenomatous polyposis coli (APC) to Asef2 is known to induce
its GEF activity; however, little is currently known about other modes
of Asef2 regulation. Here, we investigated the role of phosphorylation
in regulating Asef2 activity and function. Using high-resolution mass
spectrometry (MS) and tandem mass spectrometry (MS/MS), we obtained
complete coverage of all phosphorylatable residues and identified
six phosphorylation sites. One of these, serine 106 (S106), was particularly
intriguing as a potential regulator of Asef2 activity because of its
location within the APC-binding domain. Interestingly, mutation of
this serine to alanine (S106A), a non-phosphorylatable analogue, greatly
diminished the ability of Asef2 to activate Rac, while a phosphomimetic
mutation (serine to aspartic acid, S106D) enhanced Rac activation.
Furthermore, expression of these mutants in HT1080 cells demonstrated
that phosphorylation of S106 is critical for Asef2-promoted migration
and for cell-matrix adhesion assembly and disassembly (adhesion turnover),
which is a process that facilitates efficient migration. Collectively,
our results show that phosphorylation of S106 modulates Asef2 GEF
activity and Asef2-mediated cell migration and adhesion turnover