Nature sub-issue: Self-assembled proteins that store cell "memory" and can be "viewed" using light microscopy

As cells perform their daily functions, they turn on a variety of genes and cellular pathways
.
MIT engineers have now induced cells to record these events in a long chain of proteins that can be imaged with a light microscope
.

Cells programmed to produce these strands are constantly adding building blocks
encoding specific cellular events.
These ordered protein chains can then be labeled with fluorescent molecules and read under a microscope, allowing researchers to reconstruct the timing
of the event.

This technique can help elucidate the fundamental steps
of processes such as memory formation, response to drug treatment, and gene expression.

Edward Boyden, a researcher at the Howard Medical Institute and a member of MIT's McGovern Brain Institute and Koch Integrative Cancer Institute, said: "At the organ or body scale, there are many changes that occur over the course of
hours to weeks and these changes cannot be tracked over time.
" If the technology can extend working hours, it could also be used to study processes
such as aging and disease progression, the researchers said.
Boyden is the senior author of the study, published today in
the journal Nature Biotechnology.
Former McGovern Institute J.
Changyang Linghu, a postdoctoral researcher at Douglas Tan, is the paper's first author
.

Biological systems, such as organs, contain many different kinds of cells, all of which have different functions
.
One way to study these functions is to image proteins, RNA, or other molecules within cells, which can provide clues
to the cell's activity.
However, most methods of doing so only provide a glimpse of the moment or do not work
for very large cell populations.

"Biological systems are usually made up
of a large number of different types of cells.
For example, the human brain has 86 billion cells
.
Linghu said
.
To understand these biological systems, we need to observe the physiological events
that occur over time in these large cell populations.
”

To achieve this, the team came up with the idea of recording cellular events as a series of protein subunits that are constantly added to the
chain.
To create their strands, the researchers used engineered protein subunits, which are not normally found in living cells and can self-assemble filaments
.

The researchers designed a genetically coding system in which one subunit is continuously produced within the cell and the other is produced
only when a specific event occurs.
Each subunit also contains a very short peptide called an epitope tag — in this case, the researchers chose tags called HA and V5
.
Each of these tags can be bound to a different fluorescent antibody, making it easy to see the tag and determine the sequence
of protein subunits.

In the study, the researchers determined that the production of subunits containing v5 depends on the activation of a gene called c-fos, which is involved
in encoding new memories.
The subunits of the ha tag make up the bulk of the chain, but as long as the V5 tag appears in the chain, it means that the c-fos is activated during this time
.

Linghu said: "We hope to use this protein self-assembly to record the activity
of each cell.
It is not only a snapshot of time, but also a record of past history, just as
tree rings can store information permanently over time.
”

In this study, the researchers first used their system to record the activation of C-FOS in neurons grown in a lab dish
.
The c-fos gene is activated by chemically inducing the activation of neurons, which results in the V5 subunit being added to the
protein chain.

To explore whether this approach could work in the animals' brains, the researchers programmed the mice's brain cells to produce protein chains that revealed when the animals were exposed to specific drugs
.
Later, the researchers detected the exposure by preserving the
tissue and analyzing it with light microscopy.

The researchers designed their system to be modular so that different epitope tags could be exchanged, or different types of cellular events could be detected, including, in principle, cell division or activation of protein kinases that help control many cellular pathways
.

The researchers also want to extend the recording time
they can achieve.
In this study, they recorded events
for several days before tissue imaging.
There is a trade-off between the amount of time that can be recorded and the time resolution or frequency of event recording, as the length of protein chains is limited by the size of
the cell.

Linghu said: "The total amount of information it can store is fixed, but in principle we can slow down or speed up the growth of the chain
.
If we want to record for a longer period of time, we can slow down the synthesis and let it reach the size
of the cell in two weeks.
In this way we can record for a longer time, but with a lower
temporal resolution.
”

The researchers are also working on engineering the system to record multiple types of events
in the same chain by increasing the number of different subunits that can be merged.

Changyang Linghu, Bobae An, Monika Shpokayte, Orhan T.
Celiker, Nava Shmoel, Ruihan Zhang, Chi Zhang, Demian Park, Won Min Park, Steve Ramirez, Edward S.
Boyden.
Recording of cellular physiological histories along optically readable self-assembling protein chains.
Nature Biotechnology, 2023