WEBVTT
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Now you have seen how to cultivate brown
algae and more specifically Saccharina,
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I will show you some current downstream
resources and techniques that can be used
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today on these two models
Ectocarpus and Saccharina.
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This chapter aims to present you some
technical tools that are available on these
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two models of brown algae,
Ectocarpus and Saccharina.
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Before, you saw with Giannis and
Samuel how to produce the algal material,
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which is of course the necessary
preliminary step before any lab experiment.
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So once done, I will now show you some
specific techniques which actually are
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fairly common in animals and plants,
but used only recently in algae.
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It will be first single-cell transcriptomics,
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then techniques allowing you to meet and
approach cells more closely, meaning seeing
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them, touching them, and even disturbing
them so that their integrity is altered
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essentially by irradiation by UV light.
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So let's start. First, many brown algae
have genomics and transcriptomics tools.
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Indeed, this is not difficult to extract
DNA and RNA from these two algae,
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and protocols can be found in many
publications relative to these two algae.
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They require some adjustments because
algae like Saccharina contain a high
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amount of sugar that needs to be
removed using chemicals like CTAB.
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I already gave some data about the genome
and the number of genes of these two algae
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in the previous chapters. That can be useful if
you go for genomics and transcriptomics studies.
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But genomes reflect the potential of an
organism, not its activity at one given time
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or in one given tissue or organ.
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That's what transcriptomics does,
provided that you can be precise
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enough in both the spatial
and temporal scales.
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To increase the spatial scale, we use
what is called single-cell transcriptomics.
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There are different methods to achieve
single cell-transcriptomics, and many involve
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sorting cells labelled with a fluorescent
molecule prior to RNA extraction and
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sequencing. In Ectocarpus here, we use laser
capture microdissection, abbreviated LCM.
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It consists in dissecting a piece of tissue
or even one single cell from the whole
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brown alga, usually an
embryo or a small piece of it.
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The material has to be chemically fixed
beforehand in order to keep the RNA intact.
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Then the piece of material is collected in
an Eppendorf tube from which RNA will
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be extracted and eventually sequenced.
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So now we can get information as to where
genes are expressed within an organism
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at a scale as low as the cell itself.
Can we also map the product of
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these genes with the same resolution?
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For that, we can use several techniques.
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But first, before looking for molecules in
detail, one might want to have a better
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idea of how our system, here
a tissue or a cell, behaves in time.
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We use time-lapse microscopy to monitor
how our brown algae, both Ectocarpus
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and Saccharina, grow over time.
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That allows us to capture how fast cells
grow and how fast they differentiate,
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and how fast and where they branch.
Then we can use in situ hybridization
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to work on the spatial resolution of
where one given RNA is expressed.
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You have seen this technique in many other
modules as it is commonly used in animals,
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but in brown algae the
first report is very recent.
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It allowed to detect and map RNA from
the Bromoperoxydase of Ectocarpus
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when it is infected by Maulinia.
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Polysaccharides can also be mapped by
using either antibodies raised specifically
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against epitopes of some
chosen cell wall polysaccharide.
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In this picture, cell wall polysaccharide
alginate has been detected in green fluorescence,
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while the autofluorescence of
the chloroplasts is seen in red.
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But labelling can also be done by using
chemicals or drugs. Cellulose, another
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polysaccharide of the cell wall appears here
in green fluorescence when in contact with
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calcofluor, here again in Ectocarpus.
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And actin filaments can be labelled with
a drug from a fungus named phalloidin
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which sticks to actin protein
and disturbs its dynamics.
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When fused to a red fluorescent molecule,
here the rhodamine seen in yellow,
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it allows to display where the actin
filaments are localised within each cell.
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And the blue dots are the nuclei, labelled
with DAPI and other chemical compounds
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sticking to DNA. This technique is
called immunocytochemistry (ICC)
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or also immunolocalization.
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Atomic force microscopy is a technique of
nanoindentation where a cantilever pushes
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on the surface of a cell to assess
its stiffness or even its stickiness.
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The forces necessary for the tip of the
cantilever to penetrate or to go away from
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the surface is calculated from the deviation
of a laser light hitting the surface of the cantilever.
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Using this technique, we measured the
stiffness of Ectocarpus cell surfaces,
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here of an apical cell, expressed in
pascals as indicated in the color scale
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on the right-hand side of the photo.
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But the most powerful techniques are
those able to interfere with the activity,
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the normal functioning of a cell. It can be
done at several levels, and lasers actually
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perform pretty well for that.
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First, it can simply disrupt the cell surface
so that the cells burst and eventually die.
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The technique is called laser ablation and
we used it recently in Saccharina embryos
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to study cell fate.
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UV light, when it irradiates the nucleus,
makes mutations in the DNA strands.
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That generates mutants and we carried out
such experiments in Ectocarpus several years
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ago when studying its development.
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We first screened for irradiated algae,
which displayed a strange, abnormal
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morphology, also called phenotype,
thinking that some genes involved in
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the development must have been mutated.
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Then using molecular and genetic
techniques, we identified the mutated
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genes responsible for the phenotype.
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Finally, lasers can also be used to measure
the dynamics of some cellular compartments.
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For example, the Golgi apparatus
or any other compartment that is
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regenerating itself pretty fast.
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The principle is to photobleach a cellular
component or compartment which is
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bound to a fluorescent molecule, and then
measure the time necessary for seeing it again.
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When it's done, that means that the bleached,
damaged component has been renewed by
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non-bleached material present in the
surroundings of the bleached area.
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By measuring the duration of this
process, one can quantify its dynamics.
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We used this technique to assess the
dynamics of membrane trafficking at
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the surface of the apical cell
of the filaments of Ectocarpus.
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So we have seen a few techniques allowing
you to interfere with the cellular activity
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which can be useful for any
type of research project.
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We also have seen how specific molecules,
RNA, proteins, and polysaccharides could
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be mapped within cells, helping us have
a clue of their roles in cellular activities.
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I hope that now you have a better
idea of how far you can go in your project
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and how you can find
answers to your questions.