(gentle instrumental music)
- Oh my gosh, I'm totally embarrassed.
Thank you so much Dr. Fisher.
Really great to be here.
So my name's Peter Shearstone.
I'm the Vice President of Global Quality
and Regulatory for Thermo Fisher Scientific.
We're in Waltham, Mass, and all over the world,
actually 314 different plants.
So I handed my card out to a bunch of folks earlier today,
but if I didn't get to see you,
please stop and say hi afterwards.
We're looking for good employees and interns,
depending upon your year in the university.
I'm really pleased today to introduce Jason Brown,
Dr. Jason Brown at the Darwin Festival.
I was here, where you're sitting, back in 1985.
And the big topic then was AIDS of course,
and now we know that we can treat AIDS
and I think about web year presentation today
and how fantastic it would be that maybe not 35 years later,
maybe it's five years from now,
we'll be talking about a cure for ALS, right?
Amazing presentation earlier.
Along with another amazing presentation is Dr. Jason Brown.
I'm gonna introduce Dr. Brown.
Dr. Brown earned his BS in genetics
and a PhD in cellular biology
from the University of Georgia, go Bulldogs, go Dogs.
Taught biology at Young Harris College for seven years.
He was a post-doc associate
at the University of Massachusetts Medical School.
He's been a part-time lecturer
in the Department of Biology at Tufts, Jumbos?
So you've had Bulldogs, Jumbos,
UMass is the...
What is UMass's mascot?
The Vikings?
Oh, Salem State's Vikings.
UMass.
Minute Men, I think it's the Minute Men.
Minute Men, yes.
Dr. Brown's been at Salem State since 2014
and during the fall of 2020 he was tenured
and promoted to Associate Professor.
Dr. Brown's research applies genetics, biochemistry,
and cell biology to study the assembly
and function of cilia.
These hairlike organelles,
and Anne Barker's gonna get an education here
in what hairlike organelles are,
extend from the surface of almost every cell
in the human body,
and are required for processes
to drive diverse processes such as respiration,
reproduction, vision, and embryonic development.
Dr. Brown will discuss cilia assembly,
the ciliopathies,
and the work he and his students have been doing
to understand the mechanism of cilia gene regulation
in his talk.
And his talk is sponsored by the biology department
in Thermo Fisher.
So I'll pass it over to Laura for an additional introduction
for Dr. Brown. - Thank you.
Hi.
Hello everyone, and hello to our online audience as well.
So you already heard great things about Dr. Jason Brown
in this introduction
and you might be impressed even before he starts speaking.
Well, he has over 22 years of teaching experience,
and when I was talking to him about that,
he said that it made him feel really old.
With that being said most of our students
are younger than 22, so,
so his mentoring experience goes back all the way to 1999,
and his honors and awards started in 1993,
when he was a presidential scholar at UGA.
That's a fun fact for you guys.
He has been investing in his teaching
by attending multiple meetings and workshops,
and his scholarly success is demonstrated
with over 16 publications.
With all this success,
you who might not know him in person,
might feel a little intimidated,
and I'm here to share with you a few words about him
in addition to the accomplishments on his CV.
Dr. Brown is a great colleague.
He cares for his peers and students,
and he is always trying to help everyone around him.
He comes in early, leaves late,
and always has time to lend a hand.
He's respectful and respected, sympathizes with others,
and many times has worked above
and beyond his required job description to help students
and faculty who are still learning the ropes, like myself.
In the few years I have known him,
I have seen firsthand how well he collaborates, teaches,
learns, and most importantly, focus on his goal,
which is improving learning in the classroom and lab.
It is a great honor to introduce you to my colleague,
Dr. Jason Brown.
- Let me get these things outta the way.
Thank you so much for the introduction. Wow.
So,
thanks to the biology department
and to the Darwin Festival committee
for inviting me to speak.
Thanks to Thermo Fisher Scientific
and the biology department for sponsoring the talk,
and thanks to you all for being here
and the online audience as well.
So when I was thinking about preparing this talk,
I thought a lot about what it means to do research
at a primarily teaching oriented institution.
And for me, it's really about the students.
And so in addition to, I'm talking to you about cilia today,
I want to talk to you about experiences
that I've had over the years
with a number of
really outstanding undergraduate researchers.
And so we'll start out talking about the significance
and structure of cilia.
I'll talk to you about some,
a couple of really important research questions
in this field,
namely how are cilia assembled?
And how is the motility of cilia generated and regulated?
And then really the bulk of the talk is gonna be
three kind of vignettes about undergraduate researchers.
Sort of snapshots of experiences
that I've had with undergraduate researchers over the years,
including web, in the back of the room, so.
So cilia were first described about 345 years ago
when Leeuwenhoek using really high quality microscopes
that he had made himself,
saw single celled eukaryotic organisms under the microscope,
and he described those as animalcules,
and the cilia that he saw in those organisms
as these incredibly thin little feet
or little legs.
The cilia that you're seeing
at the bottom of the screen here
are actually at ependymal cilia,
which extend out from glial cells
in a mouse brain in this case.
And this is work from Karl Lechtreck
working in George Witman's lab
at University of Massachusetts Medical School,
when he was there.
He's now at the University of Georgia, Go Dogs.
And then it was actually almost exactly 300 years later
when the first disease caused by immotile cilia
was described by Afzelius.
And the patients with this immotile cilia syndrome,
have chronic sinus and respiratory infections.
They're infertile, and kind of amazingly,
about half of them have something called
Situs inversus totalis.
And this is a rare condition where the abdominal
and thoracic organs are in a mirror image position
relative to the normal positions of the organs.
So in 2000,
the field or the connection between cilia and diseases
really started to take off as an interesting research area
when this paper was published
looking at a mouse model of polycystic kidney disease.
So PKD effects somewhere between one in 500
and one in a 1000 live births.
And people who have polycystic kidney disease
typically need to go on dialysis
or end up needing a kidney transplant.
So on the right, you're looking at a normal human kidney
in the center,
and these are kidneys of people
with polycystic kidney disease.
So you can see it's a really dramatic effect on the kidneys.
In this same study,
they found that the gene that was mutated
in their mouse model of polycystic kidney disease
was this gene Tg737.
And the other thing that they found was that a mutation
in the homologous gene to that one
in this single cell organism,
Chlamydomonas reinhardtii,
caused this organism,
which is a single cell green alga, to lack its cilia.
And so of course they immediately went
and looked in the kidneys in their mice
to see if they saw any cilia defects.
And what they found was,
inside the kidney collecting duct tubules,
there were cilia extending out from the surfaces
of the cells in that tubule.
But in the mutant mouse,
the cilia were either completely missing
or very much reduced.
So at this point,
researchers got really excited
about the possibility that there might be other diseases
that are caused by problems with cilia,
other than just immotile cilia syndrome.
And one of the reasons
that they got so excited about this,
is that cilia are found on virtually every cell
in the human body.
So they're not found on red blood cells,
but basically just about every other cell in the body.
So the cilia in humans come in two varieties,
the motile cilia and the primary cilia
or the immotile cilia.
So for the motile cilia,
they include the ependymal cilia
that I just showed you a minute ago,
as well as oviduct cilia,
nodal cilia, and respiratory cilia,
which are all moving fluids across the surfaces of cells
and sperm flagella, which are moving whole cells.
The primary cilia, which are the ones that are not beating,
they actually just extend out from the surfaces of cells
and they act like a little antenna
to receive signals from other cells.
Those include olfactory cilia in our nose,
which are packed with odorant receptors
that allow the sense of smell,
and the photoreceptor outer segments in the eye,
which are packed with visual pigments that allow vision
and those kidney collecting duct cilia
that I mentioned before, among many others.
I'm just showing you a few examples here.
So it turns out that now we know that
there are a variety of human diseases
that are caused by problems with cilia.
In fact, this whole class of diseases
is now called the ciliopathies.
And ciliopathies include diseases
that are caused by problems with motile cilia.
In those cases people have infertility
and that's due to the inability of the oviduct cilia
to move egg cells and the inability of sperm cells to swim.
Also chronic sinus and respiratory infections.
And sometimes those can actually be so severe
that it causes scarring of the lungs,
which is called bronchiectasis.
And then that left right asymmetry defect
that I mentioned before,
where the organs are in this mirror image position,
that's actually caused by problems with cilia
on the embryonic node,
which is a structure on the surface of the embryo
that's involved in establishing the left-right asymmetry
during embryonic development.
Hydrocephalus is an accumulation of fluid in the brain
that's caused by a problem with those ependymal cilia
that I showed you before,
which is involved in moving cerebrospinal fluid around.
The disorders that are caused by problems
with the primary cilia include cystic kidneys, blindness,
obesity, and skeletal and nervous system abnormalities,
which are all the common theme,
they are caused by problems with signaling
that those cilia are involved in.
So one of the other things
that explains why cilia can have so many different effects
on our health is that cilia
are really complicated structures.
So they're made out of hundreds of different proteins
and they're organized though all around microtubules,
which are composed of alpha and beta tubulin subunits
that are organized into these helical filaments
that are part of the cytoskeleton in the cell.
The core of cilia is called the axoneme
and it's composed in both the non motile cilia,
which are like the ones on the top,
and the motile cilia on the bottom here,
they have nine microtubule doublets
that are arranged into a cylinder around the outside.
And in the motile cilia,
there's also this structure called the central pair,
and a number of other structures like inner
and outer dynein arms,
and radial spokes that are involved in generating
and regulating the motility of the cilia.
So on the left side here,
you're gonna see an animation
that is from cryo electrons tomography work
that was collaboration between Win Sale's lab
at Emory University
and Daniela Nicastro's lab when she was at Brandeis.
And what we're looking at is a single one of these
microtubule doublets over here,
on this side, the outer dynein arms,
and on this side over here,
the inner dynein arms,
and then these structures sticking out on this side
are these radial spoke structures
that you're looking at over here.
So we're looking at just one of these doublet microtubules
and just a very short part
of one of those doublet microtubules.
So that hopefully gives you kind of a sense
of the complexity of these organelles.
The axoneme is covered by the ciliary membrane,
which has a distinct lipid
and protein composition from the plasma membrane,
but it's connected to
and is continuous with the plasma membrane.
So there's this region at the base of the cilia
that keeps them sort of separate
and determines that distinction between the plasma membrane
and the ciliary membrane.
So really importantly that super structure
that I'm showing in there,
as well as the proteins that are found
in the cilia are really highly conserved
between different eukaryotic organisms.
And so what you're looking at here are electron micrographs
of cross-sections of cilia from human and chlamydomonas,
that single celled green algae
that I just showed you a few minutes ago.
So I hope you would agree it's kind of difficult
to tell them apart from each other, right?
So it turns out on the left is the human cilium
and on the right is the chlamydomonas cilium.
In fact,
it turns out that cilia are found
in every major lineage of eukaryotic organisms.
And what that tells us is that
the last common ancestor of all eukaryotes
was almost certainly a ciliated organism.
The other reason that's important
is because it means that we can use
a variety of different model organisms
to study cilia function and assembly.
So for the rest of this talk
I'm gonna be focusing on organisms in two clays here,
the alveolates,
which includes ciliates,
and chloroplastida, which includes the green algae.
So these are movies of these two organisms
slowed down dramatically,
so that you can actually see the cilia.
On the left is a ciliate, tetrahymena thermophila,
which is covered with hundreds of cilia.
You can see a few of them in focus
around the outside edge of the cell,
but the whole cell is covered with hundreds of cilia.
And on the right is chlamydomonas reinhardtii,
which is that green alga that I introduced you to before,
that swims through its media
by pulling itself forward
with this kind of breaststroke like motion.
And just to give you a sense
of how much we're slowing these movies down,
that's chlamydomonas swimming in real time.
So we slow them down a lot, so that you can see them.
So the two questions that I'm gonna kind of focus on
for the rest of the talk here are,
how are cilia assembled?
And how is that motility of the cilia generated
and regulated?
And both of those organisms that I just showed you
have been used to study these questions over the years.
So I'd like to start with the first of those questions,
talking about cilia assembly.
And during the process of building a cilium,
a cell needs to transcribe and translate hundreds of genes.
The proteins that are made are assembled
into many different protein complexes
and they get transported to the cilium,
and then transported into the cilium
to their sites of assembly
so that the cilia can then grow.
What we now know is that many of those proteins
are actually transported into the cilia.
Oh, let me back up and say one thing.
Cells need to have a mechanism to get these proteins
into cilia and transport it in the cilia,
because proteins are not made in cilia.
There's no ribosomes or protein making machinery
inside the cilia.
So all of those hundreds of different proteins
have to get made in the cell body
and then transported into the cilia to where they go.
So over on the right side,
you're looking at an immobilized chlamydomonas cilium,
and this process called intra flagellar transport,
which is this bidirectional movement
of protein particles from the base of the cilia,
out toward the tip.
We're gonna talk about intracellular transport more
in a couple of minutes.
This is gonna bring us
to our three undergraduate research vignettes.
So in the first of these,
I'm gonna take you all the way back to my PhD training days.
And during my PhD I studied kinesin motor proteins.
So motor proteins use the energy from ATP
to walk along cytoskeletal filaments in the cell
and when they do,
they carry cargo with them.
So in this picture from Ron Vale's lab
that they put together to illustrate this process,
this car is carrying the cargo along filament, right?
So the motor proteins do that
and they have lots of different really important functions.
So they're carrying secretory vesicles in the cell,
they're moving chromosomes around during mitosis,
and actually in the cilium,
they're causing filaments to slide against each other,
within the axoneme,
which is the mechanism for ciliary beating.
So most of the projects that I do start with mutants.
And, just to kind of orient you
to the complexity of this problem of making
and sort of isolating these mutants,
the organisms that I'm talking about,
range from having about 17,000 genes in chlamydomonas,
to in tetrahymena cells, having about 26,000 genes,
and humans kind of fall right about in the middle of that
with around 20,000 genes.
So you all probably know that a variety of different genes
will code for proteins
with different important functions in the cell,
like analyzing chemical reactions,
or regulating the expression of other genes,
or signaling from cell to cell.
And in both of those organisms,
tetrahymena and chlamydomonas,
there are mechanisms
or methods available to generate mutations in genes.
When we do that,
very often the function of proteins will be disrupted
and then we can look to see what the effect on the cell is,
to try to build a biological story basically,
and understand what the function of that protein is.
So when I was a PhD student with Jacek Gaertig
at the University of Georgia,
we created a mutant
that was totally lacking this motor protein
called Kinesin two.
And so this is in tetrahymena,
and when we did immunofluorescence microscopy,
thank you Webb for that wonderful introduction earlier,
any of you that were here for Webb's talk,
you did a great intro to immunofluorescence microscopy.
When we did immunofluorescence microscopy on these cells,
we could stain the tubulin inside the cilia with one color,
the green that you're seeing here,
and the DNA inside the nucleus in this other color, in blue.
And what we found is that wild-type cells, as I said,
are covered with hundreds of different cilia,
and our mutant was unable to assemble new cilia
during division,
but it's also unable to maintain its existing cilia.
So that was interesting and exciting,
but we got really excited and interested
when we realized that the mutant also had a defect
in cell division.
So on the left side is a normal cell dividing ,
and you can see that during division
the cell builds a new oral apparatus,
which is the feeding structure of these cells.
And what that does for us is it helps us very easily
get oriented to what the front cell and the back cell are.
So what you can see on the right side
is that mutant cells
had divided their nuclei normally, right?
So the nuclei divided as they should,
but the cell bodies weren't dividing normally.
In fact, if we let these cells grow for longer,
they would make these massive monster cells
that had multiple nuclei in them.
So they had clearly failed
to divide multiple times during that process.
So the interesting question, at this point,
we wanted to dig into the problem with cell division further
and try to understand what was happening with that.
So we used live cell imaging
to take pictures of cells during cell division.
And if you look on the left side here,
you see that from the time that wild-type cells
started making a cleavage furrow,
until they completely divided,
was only about 15 or 20 minutes, right?
We could watch this process.
But if we looked at the mutant cells,
the thing that was interesting
is that they went almost to the end, right?
The cleavage furrow almost completed, but around 25 minutes,
most of the cells,
even though they had almost finished their cleavage furrow,
hadn't completely divided yet.
If we looked at later time points,
we could actually start to see those mutant cells
fusing back together to make those monsters
that we had observed.
So the question at this point
that we thought was interesting is,
is that kinesin two motor protein that we had disrupted
directly involved in cell division somehow,
or is it a more indirect effect that we're seeing?
And so, in steps our first undergraduate,
so this is Frank Hardin,
who was an undergraduate student at UGA at the time,
who was doing a project, an independent project,
as part of his cell biology lab course.
And he decided to tackle this problem
about cell division with me, as part of that project.
And what we did is use video microscopy
of live dividing cells.
And if you see on the left side,
this is the anterior daughter cell,
almost at the end of division, it's not moving.
That posterior daughter cell that you just saw,
is spinning dramatically.
So let me go back and show that to you again.
So that cell on the left is the anterior daughter,
it's stuck in place,
the daughter cell on the right is the posterior
and it's spinning like this.
And notice that's right before the cells split.
So we hypothesized at this point
that this process that we are observing,
which we call rotokinesis,
actually helps to weaken the bridge
between the daughter cells
at the very end of cell division,
and that the problem with these cells,
with the mutant cells,
was actually that they couldn't divide
because they couldn't do that process.
So that was just a hypothesis,
but we needed to test that hypothesis, right?
So the first thing we did was to take wild type cells
and actually inhibit their motility
by putting them into a viscous,
like really thick media that they couldn't swim through.
Under those conditions, the cleavage furrow was normal,
but if you notice here, at later time points,
there was often this long membrane bridge
that was connecting the cells.
So it really did look like motility was important
for these cells to be able to divide.
But that wasn't enough, because we also said,
"Well if that's true,
then what if we just add motility to our mutants?
Can we get them to divide normally?"
So we did that,
we just put the mutant cells in a shaking culture,
and it turns out just by doing that,
we could get them to divide almost normally.
So this is the mutant with shaking,
and this is the mutant without shaking,
making those huge monster cells.
So that was, we felt like, pretty good support
for this idea that motility is important
for that organism to divide.
So just to conclude this part,
kinesin two was required for cilia assembly
and maintenance in tetrahymena.
Tetrahymena uses this highly coordinated kind of dance,
this rotokinesis process to complete cell division
and the kinesin two mutants fail to divide normally,
we think,
because they lack the ability to do that
rotokinesis process.
It turns out since then,
there have been a number of different papers
that have been published on
motility assisted cytokinesis processes
in other types of organisms as well.
So this is not a one-off, this happens in other organisms.
So for these vignettes, I'm gonna do a little,
"Where are they now?"
For these people.
So Frank completed his bachelor's, master's,
and PhD degrees in cellular biology
at the University of Georgia,
and later went on to found
and run for several years,
a youth education program
at the Noble Research Institute in Oklahoma,
and he's now a licensing associate at Washington University
in St. Louis, Missouri.
So thanks Frank.
So for our second vignette, we're gonna switch organisms,
we're gonna switch research questions,
and we're gonna switch institutions.
So I'm gonna talk about a question
about the regulation of motility
in chlamydomonas reinhardtii that started as a project
that I was working on when I was a postdoc
at UMass Medical School.
But then that project basically transitioned to Salem State
when I came here,
and I've had a number of students work on that
over the years.
So at this point it's important to understand that
in chlamydomonas, we can generate mutants
by introducing a DNA fragment that goes in
and it lands in a random place in the genome.
So we're making random mutations by this.
And if we happen to hit a gene
that's important for the function of cilia,
we can screen out those mutants by looking for defects
in swimming or the inability to assemble cilia at all.
And when I was a postdoc at UMass,
I did a lot of this mutant screening
and I generated quite a few of these mutants.
I generated so many of those mutants
and I couldn't handle them all.
So I sent out a number of these mutants
to other labs that I thought might be interested in those.
And so one of those mutants that I sent to a colleague,
Maureen Wirschell,
at the University of Mississippi Medical Center,
was a chlamydomonas mutant
that had a slow swimming phenotype
that suggested that it might have a defect
in those outer dynein arms that I mentioned to you before.
So just to remind you about outer dynein arms,
they're attached to those outer microtubule doublets
in the axoneme.
And what they're doing is what this is showing you
on the left here,
they are on one end of the dynein,
they're attached to one of the microtubules in the axoneme,
and on the other end they're walking along
another microtubule that's adjacent to them.
So what the effect of that is,
is that the microtubules are sliding against each other
like this.
That's the mechanism behind the ciliary beating
that I showed you before.
So that movement of cilia is about sliding of microtubules
against each other.
So on the right you're seeing a schematic
of the outer dynein arm.
It's a super complicated structure just by itself.
It's made up of multiple different proteins.
It's anchored at the base
by a structure called the outer dynein arm docking complex,
which is made of three different proteins called DC1
DC2, and DC3.
And before I jump to the next slide,
I just wanna kind of orient you very quickly
to the nomenclature, 'cause it could get a little confusing.
The protein here is called DC1.
So that's what I'm showing you there.
The gene is called DCC1,
and our mutant allele that we generated
is called oda3-6.
That's just like a nomenclature thing
that we didn't have anything to do with.
We're following the guidelines for nomenclature.
So in Maureen Wirschell's lab,
our collaborator, they used western blotting.
Thanks Webb, for the introduction on that earlier too.
Basically all you need to understand
about western blotting here,
is that it's a way to look for the presence
of specific proteins.
And so if you look in the wild type cells
where we're looking for this DC1 protein,
you see that the wild type cells have DC1, right?
The mutant, which is our oda3-6 mutant over here,
is lacking that DC1 protein,
and also lacking the DC2 protein,
which is the other one here.
And that is actually very similar
to a previously identified oda mutant, oda3-1.
So that suggested that we might have a mutation
in one of those genes, maybe in DC1 or DC2.
And so what Maureen's lab did at that point
was to use the polymerase chain reaction or PCR,
to basically look at the structure of the DCC2 gene
and the DCC1 gene in this mutant,
to see if they could find the defect.
If you're not used to looking at polymerase chain reaction
or thinking about that,
all you really need to understand at this point,
is that it's a method that allows us
to look for segments of DNA
and determine whether they're there.
And so like here we're looking for this segment
that's between these two arrowheads,
which, if you are familiar with PCR,
those are the PCR primer errors.
And so what they found was, oh yeah,
and I was gonna say also this is basically like
PCR-based COVID testing, right?
Where you're looking for a sequence
of the viral nucleic acid,
by PCR in this case.
So what they found was that
when they looked at the DCC2 gene,
it was totally normal.
They didn't see any problems with that.
The DCC1 gene was almost normal
throughout the whole gene.
But at the very beginning of the gene,
there were some problems that we could see on a gel.
So in this diagram,
this is looking at the products of this PCR reaction.
So really all you need to notice
is that in this region where these dotted lines are,
there is a difference between wild type and mutant.
So here, wild type and look different,
here wild type and mutant look different.
Wild type mutant look different.
Here they look pretty similar.
There is a band about that same size,
but Wirschell's lab, they sequenced that band,
and actually found that it wasn't the DCC1 gene
in that region,
it was a non-specific PCR product.
So this is when Daniela Montes Berrueta
joined my research group,
and Daniela, her idea was to basically analyze
that mutant further
and try to get down to the molecular details
of that mutant,
and understand what's going on at the molecular level.
And her idea was to use PCR primers
that are inside that inserted DNA,
in combination with the primers
that were in the DCC1 gene flanking that insertion site,
do PCR to generate the PCR products here,
and then sequence that, determine the order of the bases
in the PCR products that she generated,
and then analyze those sequences
to try to figure out the molecular details there.
So when she did that,
she figured out that there was a 12 base paired deletion
from the five prime untranslated region of the DCC1 gene,
which is on chromosome 17.
And at that site,
the selectable marker DNA that we expected to find there,
was there.
So that was kind of good.
It told us that that probably was the site of our mutation.
But interestingly,
there's also a piece of chlamydomonas chromosome six
that was inserted in that same site.
And maybe even more interestingly,
there was a 500 base pair region
of kind of poor sequence quality.
It didn't give us great sequence information,
but what we got out of it
was that it was about 88% identical
to the human RB1 gene.
So what we think is going on there,
is that during the process of generating that mutant,
some human DNA actually got introduced
as a contaminating sequence
that got introduced at that location.
So this was important
because this is actually the first
of these oda3 mutants that had ever been characterized
at that level of molecular detail.
And Daniela ended up as a co-author on this paper
that we published on this mutant.
So that was exciting.
So in the where are they now segment,
so Daniella is now a senior research associate
in virology and infectious disease at Moderna.
And the couple of publications that you can see here
show you that she's really been kind of at the forefront
of helping to develop some of the vaccines
against COVID variants of late.
So this is gonna take us to our last of these vignettes.
And in this we're gonna talk about
some genetic techniques
that we're trying to implement,
to think about this process of cilia assembly again.
So just to remind you,
during cilia assembly there are hundreds of genes
that get turned on.
And what we're trying to answer here
is what flips the switch?
How do these genes get regulated all at the same time
so that they can build cilium?
So it turns out that chlamydomonas is a great organism
for answering this question,
because we can get these cells
to express these genes in the lab simultaneously,
in a whole population of cells,
by getting the cells to lose their cilia
and then regrow those cilia.
And we can do this really easily
with just a brief exposure to acidic conditions.
Cells drop their cilia,
and then we return the cells to neutral
and they regrow at that point,
and those genes are expressed during that time.
So you can see here
in this graph of cilia length versus time,
that these cells are able to regrow
almost full length cilia within about 90 minutes.
So it makes it really practical to do this in the lab
with undergraduate students, right?
Between classes, we can do these kind of experiments.
So during my time as a postdoc
in George Witman's lab at UMass Medical School,
I had developed a system for us to actually be able
to follow a gene expression in these cells
during the time that they're regrowing.
And so the system is based in part
on the coding region from a gene,
from a marine crustacean called Gaussia princeps
that's involved in the bioluminescence of this organism.
So it allows these cells to glow basically in the water.
And when the GLuc protein is expressed from that,
which is this Gaussia luciferase protein,
when that's expressed from that coding region,
and then you expose that enzyme to its substrate,
coelenterazine, it releases light.
And so we can do this in the lab,
and the idea was to connect up a promoter,
which is kind of a regulatory region for the gene
to that coding region for that Gaussia luciferase,
and then put that whole thing into chlamydomonas cells,
get the cells to regrow their cilia like I just showed you,
and during cilia regrowth,
those cells would produce the luciferase,
and we'd be able to detect it
as a way to look at the expression of these genes
in those cells.
So it turned out that system worked really nicely.
So if you look on the bottom here,
this is a graph of a population of cells
regrowing their cilia after de-ciliation.
And in that same population of cells,
we're looking at the luciferase activity
in cells expressing that luciferase construct.
And in green here are the mock treated cells,
so you can see they didn't lose their cilia,
and they also don't up-regulate the luciferase.
So around 60 minutes after de-ciliation,
we get this really nice peak of that luciferase activity.
So now that we had a way to look at the gene expression,
we wanted to try to use a couple of different approaches
to tackle this problem.
So the first of these is forward genetics.
And in the forward genetics approach,
it's like what I showed you before,
we randomly mutate the cell,
and then the idea was to look for mutants
that didn't up-regulate that luciferase expression anymore.
So we had disrupted that pathway somehow.
And kind of importantly, and interestingly,
those cells very often regrew their cilia really slowly.
So sometimes we could also just screen for
slow regrowth of cilia, as another thing to look for.
And because these are random mutations in the cells,
we then need to go try to figure out where the genes are.
And a couple of students that worked really hard on this,
among others,
but a couple of the students that worked really hard on this
were Ellen Acheampong and Webb Camille.
And Ellen,
during her honors thesis work
screened about 3000 randomly generated mutants.
And among those,
14 of them had this delayed regrowth phenotype
that she was looking for,
and two of them appeared to be really interesting
'cause they looked like they were signaling proteins
which could be in this pathway somehow
of regulating these genes.
But when we dug into those mutants further,
it turns out that those genes were not actually mutated
in those mutant strains.
I showed you before,
we get these complicated inserts in chlamydomonas
in these mutants,
and it turns out that that sometimes
makes it kind of difficult
to identify these insertion sites a little bit.
And Webb also did some work on this
and screened about a thousand mutants,
and when he did the PCR and sequencing on these,
basically they were in known cilia proteins,
which were interesting,
but we were pushing hard to try to identify new pathways
that hadn't been identified before,
so we didn't follow up on those mutants further.
So we don't have any like major breakthroughs
to report from the forward genetics approach at this point,
but it's still a really promising approach
and I think it could be really important.
So Ellen is now a third year PhD candidate in microbiology
and physiological systems at UMass Chan Medical School.
And those of you that saw Webb's talk earlier,
know that he's currently a fourth year MD PhD candidate
in biochemistry and molecular biotechnology
at UMass Chan Medical School as well.
All right, so we're nearly there.
In the reverse genetics approach,
the idea is we identify genes that we pick our interest in
and then we go in and make targeted mutations
in those genes.
So not random mutations, but targeted mutations.
And the idea here was that
we were gonna use either biochemical
or bioinformatics methods
to try to identify the candidate genes,
and then use a CRISPR based approach
to try to disrupt those genes.
And so it turns out that we decided
to go with the CRISPR first,
just to show that we could do it here in our lab,
at Salem State,
before we dug in, to try to identify the candidate genes.
And so a paper was published in 2020 by George Witman's lab
and some of his collaborators,
basically showing that you could do CRISPR efficiently
in chlamydomonas,
which had not been the case prior to that.
And that Gleidia Sauli,
when she joined the research group,
was gonna basically disrupt this FAP70 gene
by inserting a hygromycin resistance cassette,
or a piece of DNA that confers resistance to hygromycin
in the middle of the gene.
For any of you that don't know about CRISPR,
basically there are three parts.
There's a nuclease that makes a cut in the gene,
and then there are these two RNAs
that are involved in getting the nuclease to the DNA.
So the nuclease enzyme binds to the DNA, opens it up,
and makes this double stranded cut at that site.
So the idea in Gleidia's project was
she was gonna start with the FAP70 gene,
target the Cas9 enzyme to the FAP70 gene
to make that cut,
and then take that hygromycin resistance cassette
and add some FAP70 sequences onto the end of it,
and then get it to insert in the middle of the FAP70 gene.
We decided to do FAP70,
it's a gene that encodes part of the central pair
in chlamydomonas axoneme,
but mainly we did it because that's one of the genes
that the Whitman lab had targeted in their paper.
So she was just trying to repeat
what they had done essentially, and it worked.
That's the short story here.
Basically she tested out some of the strains
that she generated with the polymerase chain reaction,
and again,
by amplifying with these pairs of primers
in wild type cells,
she expected to get something that was about 317 base pairs,
which she saw very nicely in the wild type.
So these are wild type cells here, but in the mutant,
she expected to see either no band between those primers
or a larger fragment,
because that hygromycin piece got inserted
in the middle of the gene.
So what did she see?
With her FAP70 primers,
only four out of the 17 strains that she tested
had the normal wild type sequence.
So that meant that 13 out of the 17 strains
were either completely missing that sequence,
or had that two kilobase insert, approximately,
that she was expecting to see in the mutants.
So it really looks like that she was successful
in getting CRISPR to work at Salem State.
The mutation rate that she got was about 76%,
which is comparable to what the Witman lab had shown before.
So that was really exciting.
Gleidia is now a research technician
in the Fanning Lab in the Department of Neurology
at Brigham and Women's Hospital,
and she's working on Parkinson's disease.
And so just next steps really quickly,
a larger genetic screen potentially,
and for that reverse genetics method,
identifying genes to try to target with the CRISPR system
that we've got working now.
So either a biochemical approach
or a bioinformatics approach.
And Campbell Boisvert has been working with me this year,
since the beginning of the year,
trying to do the bioinformatics approach
to identify those candidate genes.
So I just wanted to acknowledge the long list
of other students,
and a colleague who has worked
on this project a little bit with me,
and other mentors and students,
and all of the funding for the Salem State part of this work
has come for from the MSCA Fund for Continuing Scholarship,
as well as to an SSU Minigrant
and a Flash Grant that I was awarded for this work.
I couldn't do a talk about undergraduate research
without honoring my mentors
when I was an undergraduate researcher.
Mary Case gave me my first start in a research lab
and got me going, doing research on fungal genetics.
And Michael Bender, who's now at the NIH,
introduced me to fruit fly genetics.
So I did drosophila work with him.
So thanks so much to them,
and then I'm gonna end with this slide.
I dug around looking for this, to see if this were the case.
So when Charles Darwin
was at the University of Edinburgh studying,
he did work with Robert Grant, who was a marine biologist.
And the work was to basically take a microscope
and go look at marine life in the local area.
And in March of 1827, when Darwin was 18 years old,
he wrote this in his lab notebook.
"In this species I believe I was the first to observe
both the animal and it's ciliae."
Yes.
"In most rapid movement.
By the aid of these ciliae
it could revolve in its capsule
and when freed from it move so quickly,
as to be discernible to the naked eye
at some distance.
To what animal these ova belong.
I am ignorant?"
So if you happen to decide to do undergraduate research,
if you happen to do research on cilia,
you're in really good company.
Thank you very much.
(host faintly speaking)
- [Host] A 130 people on the webinar,
and I'll read the first question,
you don't have to repeat it, 'cause I'm unmuted at this end.
And the question is basically,
do you find that being an educator and teaching classes
helps you come up with new ideas
and questions to conduct research in the lab?
- Yeah, absolutely.
Teaching genetics and teaching cell biology,
I'm all the time having to learn new things, right?
And so the types of new cutting edge things
I'm having to learn in teaching genetics all the time,
are always giving me ideas about new experiments to do.
So just very recently I've had this flood of ideas
as I was starting to work on this talk,
a lot of which have come out of genetics courses
and cell biology courses.
So definitely.
- [Audience Member] That was a great talk by the way.
- Thanks. - So,
I have a question about Boolean genetics
on mutations and genes that are known
to cause kidney diseases,
and I'm wondering what's the best avenue
(audience member faintly speaking)
in your lab.
- Tackling disease-causing genes?
Yeah, so there's the potential for that.
In the area that we're kind of focusing on now,
with looking at gene regulation,
it turns out that gene regulation pathways
are not super well conserved evolutionarily,
there's quite a bit of divergence
between different evolutionary lineages and that.
So we don't know what we'll find in that area.
Certainly I always have my eyes open
for interesting things.
Signaling pathways maybe are a little more likely
than the direct transcriptional regulators
to be evolutionarily conserved.
But yeah, I mean I always have my eyes open for that.
One of the things that I always kind of have in my mind
with the transcriptional regulation part of it,
is that even if you find things
that are only conserved in ciliates for instance,
or single-celled organisms,
there are a number of those
that are pathogens in humans, right?
That cause infectious disease.
And so finding a novel pathway
that shuts down the ability of a single-celled organism
to make its cilia, for instance,
could be a really interesting therapeutic target
for infectious eukaryotic diseases.
- [Host] We have another question actually, this is from me.
So if you could repeat it for our online audience,
knowing that we have 130, possibly, hopefully,
students online,
what do you look for in the ideal student
to work in lab with you?
- Yeah,
so I mentioned this a little bit
in my introduction to Webb earlier.
I look for students who just have sort of a gleam
in their eye when they're thinking about
and talking about science.
I can just see that.
I just know.
They have that kind of level of excitement
that I felt where I woke up
in the middle of the night sometimes
when I was in grad school and even when I was a postdoc,
I would sometimes just wake up in the middle of the night
going like, "Oh, what I'm gonna..."
Like having an idea, an epiphany about my experiment
in the middle of the night, right?
It happens, yes.
You dream about it.
And I can just sort of like...
When a student comes to talk to me,
I kind of get a sense of,
if you have that kind of level of excitement,
I'm looking for that,
I'm looking a little bit of sort of confidence on your part.
I don't mean you have to be super confident,
but you have to be confident enough
to just go to a faculty member and ask, right?
To say like, "I don't really know what research is about.
So I'm not super confident about that,
but I'm at least confident enough
to come talk to you about it and have a conversation."
I see those two things, (fingers click)
I'm good.
That's about all I need,
and we'll sit and talk for a while and so.