Examining the behaviour of fungal cells in microconfined mazelike structures - art. no. 68590U

Abstract

Filamentous fungi like Neurospora crassa are a large and evolutionary successful group of organisms that can efficiently colonise microconfined networks like soil, wood, leaf litter, plant and animal tissues. Growth of the fungus Neurospora crassa was monitored for three-dimensional interactions with artificial profiled surface and two-dimensional interactions with a patterned surface. Specific growth parameters that included branching angles and branching distances were used to measure the responses of growing hyphae to the confining features. In three dimensional microfluidic and mazelike structures, changes in the growth parameters were observed and revealed an exceptional directional memory by growing hyphae that was maintained over long distances. Comparison with data from a previous study using another species revealed that different fungal species exhibit surprisingly specific sets of growth parameters. A second set of experiments showed that Neurospora crassa had a distinct affinity to edges and tips. On a surface covered with microscale pyramids, hyphae balanced on and bridged between tips.

Full text

Examining the Behaviour of Fungal Cells in Microconfined
Mazelike Structures
Marie Held1, Clive Edwards2, Dan V. Nicolau1
1 Department of Electrical and Electronic Engineering,
Liverpool University, Brownlow Hill L69 3GJ, Liverpool, UK;
2 School of Biological Sciences, University of Liverpool,
Crown Street L69 7ZB, Liverpool, UK
1. ABSTRACT
Filamentous fungi like Neurospora crassa are a large and evolutionary successful group of organisms that can efficiently
colonise microconfined networks like soil, wood, leaf litter, plant and animal tissues. Growth of the fungus Neurospora
crassa was monitored for three-dimensional interactions with artificial profiled surface and two-dimensional interactions
with a patterned surface. Specific growth parameters that included branching angles and branching distances were used
to measure the responses of growing hyphae to the confining features. In three dimensional microfluidic and mazelike
structures, changes in the growth parameters were observed and revealed an exceptional directional memory by growing
hyphae that was maintained over long distances. Comparison with data from a previous study using another species
revealed that different fungal species exhibit surprisingly specific sets of growth parameters. A second set of experiments
showed that Neurospora crassa had a distinct affinity to edges and tips. On a surface covered with microscale pyramids,
hyphae balanced on and bridged between tips.
Keywords: N. crassa, branching, PDMS molding, microconfinement, maze
2. INTRODUCTION
Neurospora crassa is a type of red bread mold filamentous fungus, which belongs to the basidiomycetous fungi.
Filamentous fungi are of vast ecological importance (1, 2) and have a considerable economic impact because of their
exploitation for the production of enzymes and because of their threat to public health as human or plant pathogens.
Filamentous fungi fulfil a central role in most land-based ecosystems as decomposers by breakdown of organic
materials. One of the most characteristic morphological features is the formation of radially expanding circular colonies
resulting in an efficient exploitation of nutritional resources in their environment. The growth of fungal hyphae in a
relatively fixed direction (3, 4), regular branching (5, 6) and negative autotropism (7) are very important for their
invasiveness as is the excretion of an inhibitory substance of an mycelium growing on planar medium. Regular hyphal
branching is characterised by two properties (i) a constant branching angle and (ii) constant branching distances. Another
important attribute is the ability of inner- and inter-hyphal nutrient transport (8, 9) that allows hyphal extension into and
through nutrient-poor areas. Basidiomycetous fungi naturally colonise three-dimensional, microstructured and
nutritionally heterogeneous materials. The performed experiments offer two different approaches to evaluate the three-
dimensionality of growth (a) using microfluidics technology and (b) using microstructured surfaces. Both structures are
produced by replica molding of the polymer poly(dimethylsiloxane) (PDMS) which is easy to handle, fast, inexpensive
and does not require clean room facilities. PDMS is a suitable material for live-cell imaging due to its transparency, non-
toxicity (when cured over a sufficient period of time) and permeability to oxygen and carbon dioxide (10). Its surface
chemistry can be controlled, it can be reversibly deformed and sealed irreversibly or reversibly to a number of materials
(11). Due to the experimental opportunities offered by PDMS it has been a part of an enormous number of research
projects concerning the effect of confinement on microorganisms. These include cell-based biosensor (12) and tissue
engineering (13) , the manipulation of cell shape (14), function and migration (15), the examination of individual and
bulk bacterial behaviour (16, 17), diagnostic cell arrays (18) and also methods for biocomputation (19).
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VI,
edited by Daniel L. Farkas, Dan V. Nicolau, Robert C. Leif,
Proc. of SPIE Vol. 6859, 68590U, (2008) · 1605-7422/08/$18 · doi: 10.1117/12.759453
Proc. of SPIE Vol. 6859 68590U-1

Fungal hyphae extend by apical tip growth, a highly polarized process of cell extension. A popular theory about tip
expansion suggests that molecular motors transport the material for this extension as vesicles over long distances through
the hypha to the apical pole (20) and are therefore a critical factor. The secretory vesicles are destined for the
Spitzenkörper, also known as the Vesicle Supply Centre (VSC). The Spitzenkörper is a dark body in light microscopy
that is situated at the apex of the hypha and serves two major purposes: (a) it is a collection site for these wall building
cytoplasmic vesicles and (b) it is the point of departure for these vesicles as they migrate to fuse with the cell wall to
produce new cell membrane. As the hypha extends periodic branches form near the apex that themselves also extend as
new hyphae through polarized growth in fixed directions established at the branching point. There are many assumptions
about the precursors that trigger branch initiation (21-24) but their identity is still not known nor how they influence the
cell organelles to achieve branching. The branching pattern in wild-type N. crassa is mainly lateral, i.e. a new branch
extends to the side away from the median plane of the growing hypha with both of them enclosing an angle, which we
will call the branching angle below. Filamentous fungi have the ability to overcome nutrient free areas. The hypha can
extend into a region without depending on a local intake of nutrients because the colony can support it for 200-600µm.
Shepherd (9) proved that the vacuole system takes part in this inter- and intracellular material transport as an
interconnected network of tubules and round or ellipsoid vacuoles. Material transport occurs through diffusion and
peristaltic movement of tubules. These parameters enable fungi to navigate confined structures, overcome small nutrient
free areas and therefore ensure their survival.
The manner in which an amoebae like species and a filamentous fungus negotiate obstacles in confined environments
has previously been examined for (i) the rather macroscopic true slime mold Physarum polycephalum by Nakagaki (25)
and (ii) the microscopic Pycnoporus cinnabarinus by Hanson (26). Though these two organisms are very different - P.
polycephalum consists of an acellular plasmodium while P. cinnabarinus is a multicellular plant pathogenic filamentous
fungus - both have the ability to solve mazes.
3. METHODS
Figure 1. PDMS molds; (a) scheme of a maze structure, (b) picture of PDMS mold mazes
Microfabrication: The provided Silicon mold (Fig. 1) was prepared by standard photolithography techniques and deep
reactive ion etching (DRIE) using the standard Bosch recipe (26).The Silicon master was silanised with a thin layer of
HMDS (27) (Sigma Aldrich) to prevent adhesion of the polymer. The polydimethylsiloxane (PDMS) precursor and
curing agent (Sylgard 184, Dow Corning, MI, USA) were mixed in a 10:1 ratio, degassed under vacuum, poured over the
master and overcured at 65°C for at least 8 hours to ensure full cross-linking of the monomer which can have toxic
effects on the cells (28). We peeled the PDMS cast with the microstructures off the master and enclosed them three-
dimensionally by sealing them to a second unpatterned PDMS layer. The enclosed microstructures offer lateral openings
for the introduction of water and microorganisms (see fig. 1).
The soft lithography technique of polymer replica molding does not require clean room facilities and provides an easy,
fast and inexpensive way to mass-fabricate microstructures. Possible PDMS residues on the masters after peeling were
removed by immersing them in a bath of hot sulphuric acid (Sigma Aldrich, 98%, 80°C), which cracked the polymer
chains (29).
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plain agan
35
30
25
23
0lOU 200 300 400 5 600 100 800
branching dintance (pm)
50
45
40
35
30
2S
20
15
10
plain agan
010 20 30 40 50 00 70 80 90 100 110 120
branching angle (1
Fungal culture and inoculation of microstructures: The Neurospora crassa culture used was obtained from the
School of Biological Sciences culture collection of the University of Liverpool and was maintained on malt agar at 4°C.
The nutrient medium used to grow N. crassa was malt agar consisting of 1% malt extract (MERCK) and 1.5% Agar
Agar (MERCK). The channels filled with distilled water due to the capillary forces, to preserve the filling a water droplet
was placed on both lateral openings. Fungal introduction was achieved by placement of an agar plug inoculated with N.
crassa next to the lateral channel opening. The structure was enclosed in a sealed polystyrene Petri dish to retain
moisture and allow gas exchange. The fungi were grown at room temperature and images were recorded with an
inverted trinocular microscope (Motic AE21) equipped with phase contrast objectives (4x, 10x, 20x) and the USB digital
camera Motic 2300 with a resolution of 3 Megapixel.
4. RESULTS
We used “Motic Images Plus 2.0” (Motic China Group Co., Ltd.) to record and analyse the images in conjunction with
statistical analysis using “Statistica 7.1” (Statsoft Inc., OK, USA).
Figure 2. growth parameters measured:
red double arrow: distance between two branches respectively the
‘branching distance’
blue lines: enclose the ‘branching angle’
Growth Parameters: The growth parameters measured were the branching angle and the distance between branches on
growing hyphal filaments (Fig. 2). The branching angles underlie a Normal distribution whereas the branching distances
usually correspond to a Weibull distribution. The Weibull distribution is a continuous probability distribution with the
probability function:
(1)
where and is the scale parameter, is the shape parameter and is
the location parameter. The Weibull distribution is very flexible and can mimic the behaviour of other statistical
distributions such as the Normal (β = 3.2. . .3.6), the Exponential (β = 1) and the Rayleigh (β = 2) distribution.
Neurospora crassa grown on plain agar:
Figure 3. growth parameters of N. crassa on plain agar with the branching angles – Normal distribution – in the left graph
and the branching distance – Weibull distribution – in the right graph
The hyphae of Neurospora crassa grown on agar branched periodically, the majority of branching distances lay between
100−400µm with an overall average of 219µm and a most frequent branching distance of 158µm (Weibull distribution
represented in Fig. 3). These distances were high compared to the size of the microfluidic structures and in particular to
the features within. The average branching angle in young and older hyphae was 45° with a standard deviation of 16°.
This branching angle results in the most efficient exploring and exploiting of an obstacle free, unconfined environment.
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Neurospora crassa grown in 3D microconfined mazelike structures:
We observed the behaviour of Neurospora crassa in six different geometries represented in Figure 5. Confinement
changed the growth parameters in the various structures to a different extent. Images were recorded automatically and
periodically with typical capture intervals between 3 and 10 minutes. The apical growth rate was independent from the
structures but increased from 6.5±1.3µm on plain agar to 8.1±2.0µm in the networks due to the increase of temperature
caused by the constant illumination of the structures on the microscope stage.
General Observations:
Figure 4. General behaviour observed for N. crassa in microconfined structures; arrows indicate the direction from which
the hypha entered the picture: (a) hit and split: Hyphae that hit an obstacle with an angle between 54° and 90° to the wall
split into two branches that extended to each side along the obstacle trying to find the shortest way around it. (b) nestling
and corner turns: If a hypha hit an obstacle at an angle <54° it adapted to its geometry and followed the wall resulting in a
successful corner turn for 63% of the corner collisions. The picture shows a hypha that hit the wall close to the right top
corner and followed the square geometry thereby performing a 360° change in growth direction. (c) directional memory:
Hyphae remembered their initial growth direction established at the branching point after they collided with an obstacle and
followed its geometry for some time, as soon as the original growth direction was available the hypha re-adapted to it.
Figure 4 represents some general observations valid for every tested network. N. crassa showed a remarkable memory
for the initial hyphal growth direction. Some of the structures enforced variations of the hyphal growth direction of up to
180° that after release from the obstacle resulted in the re-establishment of the initial growth direction with a maximum
deflection of only ±20°. Some features in the microfluidic networks were small enough to appear as bottlenecks to the
hyphae. While passing such a structure, a branching trigger built up that induced a new filament right after passing the
bottleneck thereby offering the possibility of a spatial control of branches. Due to the confinement there were many wall
collisions that N. crassa responded to with nestling to the wall in case the collision angle was smaller than 54.3°. At the
point of collision, the hypha adapted to the wall geometry and followed it until the initial growth direction was available
again (Fig. 4(c)). At that point, the hypha eventually detached from the wall and re-established the original growth
direction. This peculiar wall attachment may have its origin in the natural environment of N. crassa, which is burned
plant matter. This material is rather porous and easy to penetrate and therefore the apex needs to be more flexible than
being able to exert high pressures on obstacles. This apical flexibility enabled the close adaption to the channel walls and
allowed hyphae to turn corners of 90° and more (Fig. 4(b)). A different peculiarity originating from the apical flexibility
is the ability to split into two individual apexes. If a hypha hit a wall, enclosing an angle larger than 54.3°, it split into
two hyphae that tried to grow around the obstacle departing from the collision point (Fig. 4(a)).
Specifics in the microfluidic mazelike structures:
N. crassa showed specific reactions to every structure resulting in individual average branching angles and branching
distances depending on the features in the networks. The “diamond” structure (Fig. 6(a)) consisted of square pillars
arranged periodically at regular distances. This structure comprised solely angles of 90° that resulted in an average
branching angle of 90.1° and a very small standard deviation of only 1.8°. Often the hyphae “zigzagged” through the
pillars, which is an expression of the flexibility of the hyphal apex of N. crassa. Another structure that comprised solely
right angles is the structure “small maze” (Fig 6(e)). However, the features were not periodic and therefore this structure
resulted in a very similar average angle (89.1°, see Fig. 7 and Table 1) but broader Normal distribution of branching
angles and a standard deviation of 14.9°. The “cellular” structure comprised branching possibilities at the angles: 30°,
45°, 60° and 90°. However, the distribution of angles did not show any peaks at these angles. The confinement caused
the branches to ‘aim’ for larger branching angles and therefore they omitted the offers and finally that resulted in the
average of 75.6°.
Proc. of SPIE Vol. 6859 68590U-4

_1
branching angles in microflu'dic structures
cellular
10 20 30 40 50 60 10 60 90 100 110 120 130 140 160 small mazes
branching angle (1
;1
p.
Figure 5. filaments of N. crassa in the different structures; the arrows indicate the respective growth direction of hyphae
entering the recorded structure
(a) diamond, (b) cellular, (c) comb, (d) stripes, (e) small maze, (f) big maze where the white line indicates how far the
hyphae explored the black-lined solution path of the big maze, (g) roundabout structure
The “stripes” structure consisted of parallel straight channels with varying width (between 10µm and 2µm). Branching
occurred in channels that were wider than the diameter of the hyphae, which usually attached to one side of the channel
with an increased number of branches emanating from the nonconfined side. N. crassa grew through every channel
width whereas for narrow channels, the hyphae first grew into them and when the hyphae became more mature parts
distal from the apex expanded the channel width. This is a favourable behaviour to break down matter mechanically and
subsequently exploit the nutrient sources. The “big maze” structure had an edge length of 1mm and the individual
channels were 20µm wide, which meant less confinement for the hyphae. The distribution of branching angles shows
two peaks (i) at 47° that is close to the branching angle without confinement and caused by the freedom in the wide
channels and (ii) 85.2° that is caused by the solely 90° corners of the features.
Figure 6. Normal distribution of branching angles in mazelike structures
branching angle (°) structure
Normal Distribution
average
plain agar 45 ±16.1
diamond 90.1±1.8
cellular 75.6±16.4
stripes 80±22.2
big maze 68.9±21.7
small maze 89.1±14.9
Table 1: average branching angles and
standard deviations of N. crassa in different
mazelike structures
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branching distances in micrafluidic sftuctures
0 50 100 150 200 250 300 350 400 450 bigmaze
branching distance (pm)
The branching distances show that confinement has a vast influence (Fig. 7 and table 2). The Weibull branching distance
maximum in the structure “small maze” decreased to a value 16 times smaller than the branching distance on plain agar.
The mean free path of hyphae was smallest in the “small maze” structure and therefore resulted in the most vigorous
impact. Surprisingly, the “stripes” structure that consisted of parallel channels resulted in a lower branching distance
because hyphae usually hit a channel partition wall and split before entering (Fig. 6d). Additionally, if the channel width
was smaller than the hyphal diameter a branch emanated directly after the apex left the confinement. The branching
distance in the “diamond” structure was determined by small multiples of the pillar distance and is the second lowest
value after the “small maze”. The mean free path in the structures commonly influenced the branching distances, which
reflects in the distributions in Fig. 7 and Table 2. The periodic “comb” structure suppressed branching completely.
Hyphae adapted very well to the periodicity but they did not form branches at all. After the hyphae left the periodic
structures, we observed a unique behaviour: up to the first two branches emanated after very small distances and
subsequently the distances were larger, i.e. the hyphae seemed to recover from the periodic confinement. The change in
branching distance was not gradually but rather abrupt and this kind of branching pattern was just present for this
particular structure. According to Watters (23) there might be two distinct origins of branching precursors, the tip itself
and the colony. The information from the tip that is less confined after leaving a structure differ from those stemming
from distal, more mature hyphal areas that are confined in the periodic channels. The influence of information from
distal parts decreases with the apical extension, which could possibly explain a general recovery process but questions
remain: Why does the influence drop abruptly and why does it happen for only one of the examined structures?
Figure 7. Weibull distribution of branching angles in mazelike structures
Species Comparison:
feature Neurospora crassa Pycnoporus cinnabarinus (26)
branching rates increase depending on the
confinement but in narrow periodic
structures that suppress branching
increased almost twice in small
mazes
branching angles 45°±16° on plain agar;
increased angles within the structures,
depend on the specific properties, e.g.
89.1°±14.9° in small mazes
79.8°±29.9° in small mazes
wall collision nestling to the wall as long as it
obstructs the original growth direction
‘nosing’ along the wall in the
direction closest to that of original
growth
directional memory extraordinary memory, hyphae turn
back toward their initial growth
direction even after structure enforced
directional changes of up to 180°
hyphae turn back toward their initial
growth direction when the structure
geometry allowed them
branching distance
(µm)
structure
Weibull Distribution
maximum
plain agar 158
small maze 9.9
cellular 18.1
stripes 24.3
big maze 36.9
diamond 14.9
Table 2: maximum of the Weibull distribution
of the branching distances in different
mazelike structures
Proc. of SPIE Vol. 6859 68590U-6

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apical growth rate unchanged by confinement unchanged by confinement
corner response the hypha turns a corner in 63% of all
cases, does not turn and stops growth
or squeezes between the PDMS layer
in remaining 37%;
rare initiation of subapical branches
after collision
in 21% the hyphae turn to follow the
geometry, in 79% they continue
apical extension into the corner,
causing the hyphae to bend;
initiation of subapical branches
when the hyphae do not turn
u-turns possible for individual hyphae when
enforced by the microfluidic
structure; largest angle a hyphae turns
in small scale confinement: 281°,
large scale confinement: 360°
93-94° is the hyphal turning limit
hyphal flexibility apex is very flexible resulting in the
hyphae to attach to the walls and
therefore adapt to the geometry, the
established hyphae are rather stiff
the apex and the hyphae themselves
are quite stiff
Table 3: summary and comparison of the behaviour of N. crassa and P. cinnabarinus in microconfined microfluidic
networks
Neurospora crassa grown on 2D PDMS arrays:
The second set of experiments took place on patterned surfaces that were not enclosed in the third dimension. The
structures were again PDMS molds casted from Silicon masters (1cm×1cm) and contained square arrays of 25 cylinders
(with a rounded top) or pyramids with a height of 5µm and varying density. The experimental layout was as follows: the
PDMS surface sat between two blocks of agar - one of them was inoculated with N. crassa - with the arrays facing
upwards and no confinement in the z-direction. The colony grew in a closed Petri dish for 1-2 days at room temperature
and hyphae explored the patterned PDMS. N. crassa showed no general tendency to accumulate at the structures or to
avoid them, neither at the low-density nor at the high-density patterns. Both cylinders and pyramids acted as obstacles
and could ‘guide’ the hyphal growth through the arrays.
Figure 8: example for a hypha growing on top of pyramids; the left image is recorded with the focal plane at the surface and
the right image with the focal plane on the hyphae on top of the pyramids
However, there was a distinct difference between the guiding effect of cylinders and pyramids. In contrast to the
cylinders, the hyphae attached to the edges and corners of the pyramids basements whereas they did not attach to the
cylinders at all. We already observed this particular affinity to straight walls in the microfluidic structures. It seems there
is not only an attachment due to the frequent wall collisions resulting from the spatial restriction but also a general
affinity to straight and corner features. The natural environment of N. crassa is burned plant matter that is very porous
and brittle. Therefore, it is favourable for the hyphae to attach to structures that stabilise the system and the colony.
Another surprising behaviour that occurred at the pyramidal patterns is that some of the hyphae grew on the tips of the
pyramids. In some cases, the arrangement of pyramids could even dictate the path of the growing hypha. The distance of
the pyramids is a critical parameter for this process (Fig. 8). For the patterns with the highest density there was no hypha
Proc. of SPIE Vol. 6859 68590U-7

growing on the tips just in between the basements though these structures are not omitted in general. The question if a
hypha could bridge between two pyramids depended on the stiffness that could result in sagging and attachment to the
surface in between the pyramids if the distance was too large. If it was small enough, there was no sagging. Neurospora
crassa is a fungal species that grows according to negative autotropism, i.e. it grows opposite to the gravitational force.
Additionally, the PDMS surface in the experiments was nutrient free and the hyphae starved while exploring it. We
assume that these are the main reasons that the filaments tended to hover over the surface and balanced on the tips of the
pyramids. Some hyphae seemed to use the pyramids as a lift-off ramp. In the pictures represented in Fig. 8, it appears as
if the hyphae climbed up and down the pyramids along one of the four edges growing diagonal respective to the square
base. In contrast to the pyramidal arrays, no hypha grew over a cylinder. Therefore, it becomes clear that N. crassa is
very much attracted by sharp tips in contrast to round structures. Occasionally, we could observe that the apex of hyphae
that grew up a pyramid ‘nosed’ for another pyramidal tip top attach to.
5. DISCUSSION AND FUTURE WORK
The experiments we performed are similar to a previous study of another fungal species that introduces a methodology
comprising both experiments and simulations (26) for testing the fungal space searching ability. However, the results of
both experimental studies are surprisingly different. The growth parameters, i.e. branching angles and branching
distances, of N. crassa change to a larger extend due to the confinement and it shows some very different general
behaviour than P. cinnabarinus (26). Future stochastic simulations will show which species applies algorithms that are
computationally more efficient to solve the given mazes. Different natural fungal strategies may therefore help to solve a
larger variety of non-trivial geometrical problems. A more comprising set of algorithms might be accomplished by an
evaluation of the algorithms of a variety of fungal species, which can lead to some global rules. The innercellular
parameters and mechanisms involved in information processing, growth and branch formation are still not known but
necessary to understand the full impact of the confinement and the change in growth parameters. This raises many
questions: What are the cellular triggers for branches and how do they change the branching distances and angles in
confinement? Which are the important cell organelles, e.g. cytoskeleton, vesicles, Spitzenkörper, and their roles in
branch formation? What happens to the Spitzenkörper when a hypha hits a wall and splits into two new branches?
Neurospora crassa is an excellent species to observe because the genome is fully known and there is a variety of mutants
that have defects in morphology and growth behaviour and many chemical agents that can influence cell organelles and
therefore the growth behaviour. We want to test the behaviour of selected mutants or chemically changed hyphae in the
networks to maybe find an answer to some of the questions above. Furthermore, we will observe the innercellular events
with the help of fluorescent and confocal microscope techniques.
The particular affinity of N. crassa to edges and corners offers the possibility to guide the growing hyphae more gentle
than microfluidic networks but efficient and without causing a vast change in growth parameters. We aim to find the
optimal size and distribution of pyramids and integrate these structures in a three-dimensional assembly to reduce
disturbing influences. Subjects of particular interest are the hyphae on top of pyramids. Does the pyramid tip penetrate
into the cell wall and how deep does it penetrate? What happens with the cell organelles ? How does a hypha find a new
pyramid tip to form a bridge and what happens inside the cell during these processes?
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