WEBVTT
1
00:00:05.570 --> 00:00:10.449
Welcome to the final chapter on
hot research topics in brown algae.
2
00:00:11.128 --> 00:00:14.852
This chapter will show you some recent
results obtained by academic research
3
00:00:15.152 --> 00:00:21.482
in Saccharina or Ectocarpus, and I chose
some examples which highlight the diversity
4
00:00:21.782 --> 00:00:25.444
of mechanisms due to the
specific evolution of brown algae.
5
00:00:26.494 --> 00:00:30.985
But I also chose some examples which
show, despite this specific evolution,
6
00:00:31.285 --> 00:00:36.420
that this organism has selected
mechanisms common to animals and plants.
7
00:00:37.340 --> 00:00:43.316
Altogether, this chapter will again illustrate
the high diversity of mechanisms that these
8
00:00:43.616 --> 00:00:49.546
organisms display, and more importantly,
that a great deal of knowledge still needs
9
00:00:49.846 --> 00:00:53.250
to be discovered and they
are all waiting for you.
10
00:00:54.100 --> 00:00:59.140
This chapter will illustrate some academic
research topics currently carried out in brown algae.
11
00:01:00.400 --> 00:01:04.485
Because the aim of fundamental research
is to understand how organisms live,
12
00:01:04.685 --> 00:01:08.570
research focuses on several levels
of what makes a brown alga.
13
00:01:09.390 --> 00:01:10.700
First, the cell.
14
00:01:11.870 --> 00:01:15.968
Topics focused on the formation and
composition of the cell wall, on the
15
00:01:16.168 --> 00:01:21.961
organisation of the cytoskeleton, and on
vesicle trafficking are explored mainly
16
00:01:22.161 --> 00:01:23.510
with microscopy techniques.
17
00:01:24.360 --> 00:01:28.240
The factors involved in the dynamics of
cell division used to be studied mainly
18
00:01:28.440 --> 00:01:29.830
in the brown alga Fucus.
19
00:01:31.204 --> 00:01:35.675
And this leads us to the formation of tissues
where several cells communicate together
20
00:01:35.875 --> 00:01:40.320
to organise themselves in a functional
group of cells with specific functions.
21
00:01:42.000 --> 00:01:46.150
While the alga grows, it interacts with
the other organisms sharing the same
22
00:01:46.450 --> 00:01:51.351
environment and this can be microorganisms
like bacteria or fungi, either pathogens or
23
00:01:51.551 --> 00:01:57.010
symbiotes, or other ones like
metazoans and even other algae.
24
00:01:57.910 --> 00:02:02.050
Knowing how brown algae reproduce
contributes to study the dynamics of
25
00:02:02.250 --> 00:02:06.800
their population along the marine
coast and to anticipate future changes.
26
00:02:08.930 --> 00:02:13.160
In this chapter, two examples of current
research topics will be presented.
27
00:02:13.360 --> 00:02:17.659
The first one is related to the growth of
brown algae, and the second one to
28
00:02:17.859 --> 00:02:19.840
their interaction with bacteria.
29
00:02:20.830 --> 00:02:22.460
Let's start with the growth.
30
00:02:23.120 --> 00:02:26.150
It deals with the growth of
the filaments of Ectocarpus.
31
00:02:27.220 --> 00:02:31.348
We saw in the previous chapter that
Ectocarpus grows as a tuft of unicellular
32
00:02:31.648 --> 00:02:34.370
filaments, microscopic most of its life.
33
00:02:35.270 --> 00:02:40.629
What we see in these photos are sporophytes
that reach about 5mm in about 3 weeks
34
00:02:40.829 --> 00:02:42.790
when grown in Petri dishes.
35
00:02:44.800 --> 00:02:49.702
The apical cells of each filament grow and
this is what produces most of the biomass
36
00:02:50.002 --> 00:02:55.860
of the sporophyte. This is called tip growth,
shown in red in the right hand schema.
37
00:02:57.480 --> 00:03:01.312
Tip growth occurs from the very beginning
of the development of the sporophyte,
38
00:03:01.612 --> 00:03:05.953
meaning from the germination of the
zygote, as seen in the top movie, to
39
00:03:06.220 --> 00:03:08.630
branching, shown in the bottom movie.
40
00:03:10.950 --> 00:03:15.961
Tip growth is actually very localised at the
very tip of the filament. This is illustrated
41
00:03:16.261 --> 00:03:18.300
in this photo on the right hand side,
42
00:03:18.630 --> 00:03:24.410
where the cell wall has been stained with
calcofluor, seen here in green under UV light.
43
00:03:25.440 --> 00:03:30.574
After calcofluor removal, the alga was
left to grow for three hours and then
44
00:03:30.874 --> 00:03:32.360
observed under UV light again.
45
00:03:33.700 --> 00:03:38.690
The dark area on the top of the cell
displays the newly formed cell wall
46
00:03:39.250 --> 00:03:41.720
showing where growth took place.
47
00:03:43.850 --> 00:03:48.488
So the question of this research topic
is what are the mechanisms that make
48
00:03:48.788 --> 00:03:50.340
this tip growth possible?
49
00:03:51.850 --> 00:03:54.860
To answer this question, we
use the biophysical approach.
50
00:03:55.600 --> 00:03:59.485
The idea was to characterise
two main biophysical factors.
51
00:03:59.785 --> 00:04:05.383
First, what I call here the pushing force.
This is mainly the turgor in plant cells,
52
00:04:05.683 --> 00:04:08.620
but not only, as we will
see it in the next slides.
53
00:04:09.430 --> 00:04:11.850
This will make what is
called the wall stress.
54
00:04:13.790 --> 00:04:19.361
The second one is a resisting force
which relies in plant organisms almost
55
00:04:19.661 --> 00:04:23.200
exclusively on the mechanical
properties of the cell wall.
56
00:04:24.770 --> 00:04:31.650
As a result of these two opposed forces,
the cell will grow. This is called the strain
57
00:04:32.490 --> 00:04:35.000
and that's what we modelled and simulated.
58
00:04:36.220 --> 00:04:40.350
The first step consisted in measuring
several biophysical parameters.
59
00:04:41.300 --> 00:04:46.339
The first one was the turgor and we did
it using a series of solutions with different
60
00:04:46.639 --> 00:04:50.680
osmotic pressures into which
we immersed the alga.
61
00:04:52.130 --> 00:04:56.972
By finding the solutions in which
Ectocarpus' apical cells did not modify
62
00:04:57.172 --> 00:04:59.700
its volume in response to
these osmotic solutions,
63
00:05:00.310 --> 00:05:03.620
we managed to calculate
the internal turgor pressure.
64
00:05:04.540 --> 00:05:07.780
This technique is named limit plasmolysis.
65
00:05:09.190 --> 00:05:14.342
When compared to the pollen tube that
also grows by tip growth, we notice that
66
00:05:14.642 --> 00:05:16.870
the turgor is about twice higher.
67
00:05:19.320 --> 00:05:23.160
We then measure the curvature
at the dome of the apical cell.
68
00:05:23.960 --> 00:05:28.150
Indeed, the curvature of the cell is a
factor that counts in the calculation of
69
00:05:28.350 --> 00:05:33.450
the physical forces. The higher the
curvature, the lower the wall stress.
70
00:05:35.100 --> 00:05:39.830
Curvature was calculated both in the
meridional axis, meaning along the
71
00:05:40.130 --> 00:05:46.350
surface of the cell indicated by the red line
here, and in the circumferential axis of the cell.
72
00:05:47.420 --> 00:05:52.308
And what we found is that the curvature
of Ectocarpus' apical cells is similar to that
73
00:05:52.608 --> 00:05:59.588
of the pollen tube. The final biophysical
parameter was the thickness of the cell wall.
74
00:06:00.500 --> 00:06:05.679
For that, we performed transmission
electronic microscopy on longitudinal
75
00:06:05.879 --> 00:06:08.170
sections of Ectocarpus' apical cells.
76
00:06:08.870 --> 00:06:13.870
We used the image analysis software
to measure in nanometers the
77
00:06:14.170 --> 00:06:18.850
thickness, and corrected the value obtained
considering the position of the section
78
00:06:19.150 --> 00:06:23.900
within this pipe as shown in
the bottom part of the slide.
79
00:06:24.930 --> 00:06:29.082
What we see is that the cell wall in
Ectocarpus' apical cells describes
80
00:06:29.382 --> 00:06:31.530
a gradient of thickness.
81
00:06:32.370 --> 00:06:36.932
This is very interesting that the pollen
tube, in contrast, has a cell wall of
82
00:06:37.232 --> 00:06:39.171
regular and constant thickness.
83
00:06:41.240 --> 00:06:45.411
All these parameters finally allowed
us to calculate the wall stress.
84
00:06:45.630 --> 00:06:49.620
It was calculated at each
position along the cell surface.
85
00:06:50.700 --> 00:06:55.710
In these equations, P is then the
turgor, delta the cell wall thickness,
86
00:06:56.010 --> 00:06:59.770
and K the curvature, either
meridional or circumferential.
87
00:07:01.490 --> 00:07:05.649
You see that when integrating these three
parameters into an equation described
88
00:07:05.949 --> 00:07:10.220
by Hejnowicz et al, the reference is
indicated at the bottom of the slide,
89
00:07:10.840 --> 00:07:16.069
Ectocarpus has a cell wall stress profile
that is completely different from that
90
00:07:16.369 --> 00:07:17.540
of the pollen tube.
91
00:07:18.890 --> 00:07:24.490
In Ectocarpus, the stress is higher at the
tip, while in the pollen tube it is higher
92
00:07:24.790 --> 00:07:26.380
in the shanks of the tube.
93
00:07:27.220 --> 00:07:29.400
It is indicated with the green arrows.
94
00:07:30.760 --> 00:07:35.970
The value of the stress is now included in
the Lockhart equation, which relies on the
95
00:07:36.270 --> 00:07:39.720
viscoplastic properties of the
material, here the cell wall.
96
00:07:40.630 --> 00:07:45.369
The strain, which stands for the deformation
of the material, depends on other parameters.
97
00:07:45.769 --> 00:07:51.120
They are all related to the biophysical
properties of the cell wall, its extensibility,
98
00:07:51.420 --> 00:07:55.719
its elasticity and its isotropy, which
mainly relates to the orientation of
99
00:07:56.019 --> 00:08:01.490
the microfibres of cellulose. The strain is
calculated for each possible orientation
100
00:08:01.790 --> 00:08:07.172
of the deformation, both meridional and
circumferential, and the equations are
101
00:08:07.472 --> 00:08:09.690
given in the top right part of this slide.
102
00:08:10.590 --> 00:08:15.123
These equations are finally coded and
run by a computer to simulate the growth.
103
00:08:15.423 --> 00:08:19.493
Each running step corresponds to an
actual growth of 40 nanometers taking
104
00:08:19.793 --> 00:08:21.220
place in one minute.
105
00:08:21.940 --> 00:08:28.690
After 600 steps, the filament progresses
25 micrometres, which requires 10 hours.
106
00:08:29.560 --> 00:08:32.550
This is calibrated on the
real growth rate of the filament.
107
00:08:33.860 --> 00:08:38.570
On the right hand side of this slide, you
see an illustration of how each point is
108
00:08:38.870 --> 00:08:41.210
translated forward during the simulation.
109
00:08:42.480 --> 00:08:45.370
In this video you can see
the resulting simulation.
110
00:08:45.950 --> 00:08:50.960
Both Ectocarpus on the left and the
pollen tube on the right grow in parallel.
111
00:08:51.890 --> 00:08:57.580
But while Ectocarpus grows 25 micrometres
in 10 hours, the pollen tube grows the same
112
00:08:57.880 --> 00:09:02.160
distance in less than 3 minutes.
What is this model useful for?
113
00:09:02.970 --> 00:09:07.181
Once the model is set so that it is able to
simulate growth as close as possible to
114
00:09:07.481 --> 00:09:12.293
what is observed in living filaments, one
can play with different parameters to
115
00:09:12.493 --> 00:09:15.280
assess their contribution to
the growth mechanisms.
116
00:09:15.870 --> 00:09:19.720
For example, on this slide, this
is a yield threshold that is tested.
117
00:09:20.670 --> 00:09:24.841
The yield threshold is the minimal stress
that is necessary to deform the cell wall.
118
00:09:25.141 --> 00:09:32.062
The normal situation is shown in the middle
with a yield threshold value of 11.18 megapascals.
119
00:09:33.240 --> 00:09:38.042
On the left, the cell wall will deform with a
lower stress because we set a lower yield
120
00:09:38.342 --> 00:09:42.903
threshold for the cell wall. And on the
right, the cell wall will require higher
121
00:09:43.203 --> 00:09:48.190
stress before deforming because we
set the yield threshold to a higher value.
122
00:09:49.510 --> 00:09:55.138
You see that if the yield threshold is lower
then growth is faster, but the cell shape
123
00:09:55.438 --> 00:10:00.910
is modified to produce a cell more round
and less polarised than in the control case.
124
00:10:02.210 --> 00:10:06.760
These results give us a suggestion that
the cell shape and the polarised growth
125
00:10:06.960 --> 00:10:08.490
depend on this parameter,
126
00:10:09.200 --> 00:10:13.950
that we can then investigate with
biological techniques back in the lab.
127
00:10:14.670 --> 00:10:19.060
In conclusion, this work showed that
Ectocarpus, as a strategy, opted for
128
00:10:19.260 --> 00:10:23.141
growth completely different from that
of the pollen tube on most of the other
129
00:10:23.341 --> 00:10:24.920
tip-growing cells, actually.
130
00:10:25.630 --> 00:10:28.970
It controls tip growth through a
gradient of the cell wall thickness.
131
00:10:29.550 --> 00:10:34.450
The cell wall is very thin at the tip, less
than 10 nanometers, and it thickens on
132
00:10:34.750 --> 00:10:39.500
the shanks of the cell to reach 500 nano-
metres as the cell keeps growing forward.
133
00:10:40.500 --> 00:10:45.021
In contrast, the pollen tube controls tip
growth through the stiffness of its cell
134
00:10:45.221 --> 00:10:48.920
wall that is softer at the
tip and stiffer on the shanks.
135
00:10:50.370 --> 00:10:55.260
From a biophysical point of view, these are
fundamental differences as Ectocarpus acts
136
00:10:55.460 --> 00:11:01.380
at the level of the stress that is independent
of the mechanical properties of the cell wall
137
00:11:01.760 --> 00:11:05.299
while the pollen tube targets what
I called in the beginning of the talk
138
00:11:05.499 --> 00:11:10.690
the resisting forces that are due to the
mechanical properties of the cell wall.
139
00:11:12.150 --> 00:11:17.270
To achieve this work, we used mainly
microscopy tools, both optical and electronic
140
00:11:17.940 --> 00:11:22.300
and computer sciences to code the
model before running the simulations.
141
00:11:22.680 --> 00:11:26.050
We also needed to master
the cultivation of Ectocarpus.
142
00:11:26.350 --> 00:11:31.269
In this work, Ectocarpus grew in Petri
dishes. But since, we have developed
143
00:11:31.569 --> 00:11:36.018
microfluidics to allow the monitoring of
several filaments in parallel channels
144
00:11:36.318 --> 00:11:39.900
easing quantification of the
growth rate for example.
145
00:11:41.120 --> 00:11:45.630
The second topic of current research is
the interaction with the microbiome,
146
00:11:45.830 --> 00:11:50.130
and this will be presented to you through a
video done by Bertille Burgunter-Delamare
147
00:11:50.430 --> 00:11:52.750
a PhD student in the Roscoff lab.