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\begin{document}

\title{Bio 200C Assignment Bhumil Patel}



\author[1]{Bhumil Patel}%
\affil[1]{University of California, Santa Cruz}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\textbf{Review: Optimized FRET pairs and quantification approaches to
detect the activation of Aurora kinase A at mitosis.}

In this manuscript, Bertolin et al. improve on their original Aurora
Kinase A biosensor to produce a second generation that would help follow
AURKA activation in regions where it is extremely low in concentration
and undetectable with the original AURKA biosensor. The authors develop
two independent strategies to improve on their previous work. First,
they develop a single-color AURKA biosensor for multiplex FRET and
second, a method to observe and quantify FRET efficiency in areas with
very low AURKA abundance. The authors show that dark acceptors ShadowG
and ShadowY allow for single-color FRET/FLIM measurements while first
generation tandem GFP isn't suitable due to low concentration of AURKA.
They also show the inability of the original construct to measure FRET
by 2c-FCCS and thus develop a novel method by replacing the
donor-acceptor pair with a mTurquoise2 and novel superYFP. The
experiments allowed the authors to develop guidelines when making new
FRET biosensors such as characterizing the nature of the protein and
making sure the conformational changes of the protein fall within the
Forster's radius of the donor-acceptor pair.

The improvements to AURKA biosensors represent a novel way for studying
the function of this kinase. While fluorescence anisotropy has been used
in the past to study FRET in different kinases such as PKA, ERK, and
cAMP, it has not been known to work with AURKA due to the nature of the
protein and it's function. Also, given the fact that levels of AURKA is
regulated throughout the cell cycle, the ability to detect it at low
levels will help understand it's function in diverse contexts.

The authors provide good explanations with regards to the anomalies seen
in their data and point out any results that deviate from their expected
hypothesis. However, experiments with regards to characterizing the
effects of inserting a novel superYFP on the cell and AURKA's function
need to be seen. The author's also fail to provide clear explanations
for discrepancies between the inactivated kinases in Fig. 1B and 1C. The
author's work is systematic, giving context when constructing new
strains, and provides clear explanations when talking about new methods
of quantifying FRET. One thing that I did have a hard time understanding
was the use of anisotropy to measure FRET, and I think the authors could
have done a better job introducing the concept.

In terms of experiments that need to be done in order to further
validate the results. As mentioned previously, the differences observed
in \selectlanguage{greek}Δ\selectlanguage{english}lifetime for inactivated ShG-AURKA-mTurq2 and ShY-AURKA-mTurq2 need
to be investigated or explained better. Similarly, effects of inserting
flanking donor-acceptor pairs on the function of the kinase need to be
quantified. It would be relevant to see how insertion of the flanking
pairs affect AURKA localization to the spindle poles and morphology of
the cell compared to wildtype. It would also be interesting to see if
normal, non-arrested cells can function properly for multiple
generations with the inserted constructs.

There are minor spelling mistakes that can be attributed to continental
differences. But for the most part, the article is easy to read and well
written, however, explaining the thresholds in Fig. 1 and 2 will help
the readers. As someone who is not familiar with analyzing fluorescence
data, I did have a tough time understanding Fig. 3 and 4C, but the data
and the author's interpretation are clear and convincing.

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