Introduction the cytoskeleton system: Microtubules, Microfilaments and

Introduction

The cytoskeleton was found very late until the use of glutaraldehyde at room temperature in 1960s, which
make it observable with the electron microscope. It consists of a structural
system called cytoskeletal system, and it is named three intracellular systems
together with intracellular genetic systems and cell membranes system. In most mammals, three kinds of filaments in cells make up the
cytoskeleton system: Microtubules, Microfilaments and Intermediate filaments (Figure
1). Then dimension of microtubules is normally 25nm, the largest one, which
takes longest time to growth and it is comparatively steady than the others.
And microtubules build the main structure of cytoskeleton, supporting the
microfilaments and intermediate filaments. Microfilaments have the smallest
dimension (about 7nm) among them, and mainly make up from actin, so are named
actin filaments sometimes. The active actin filaments are formed with a complex
cycle, including many cooperation of various types of actin proteins. The
formation is assisted by a wild range of actin-binding different proteins and
sometimes overlapping activities (Jockusch, 2017). The diameter of
intermediate filaments is between microtubules and intermediate filaments,
commonly 10nm. The cytoskeleton’s varied functions depend on the behaviour of
three families of filaments, such as transformation, motility, growth and
differentiation (Alberts, 2008).

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Fig. 1.

Diagrams of the cytoskeleton system

The cytoskeleton supports the cell structure and
determine the shape. It not only determines the mechanical behaviours and stability
of cells, but also controls the motility and deformation of cells and help
cells to perform various biological functions such as transport of biological
molecules.

Recently, many important events about cell in
different time and space scales have been discovered, including the cellular
mechanotransduction. Briefly, the
cellular mechanotransduction is a big discovery in the biology, and analysis of
this subject need to understand ‘Stretch-activated ion channels, caveolae,
integrins, cadherins, growth factor receptors, myosin motors, cytoskeletal
filaments, nuclei, extracellular matrix, and numerous other structures and
signaling molecules’ (Ingber,
2006).
And it is a process that outside signal transmits to intracellular components
while cell contacts the environment, which influences the growth/depolymerisation
and cross-linking/unbinding of cytoskeleton. This process consists of a range
of dynamic response. In contrast, this dynamic response will make an effect on
the environment and both of them achieve a dynamic balance eventually. Cellular
mechanotransduction has a deep influence on cell and biodiversity, and
exploring and discovering cellular mechanotransduction not only promote the
development of biomedicine, but also like the goal scientists want to achieve. The
study in cellular mechanotransduction still has numbers of problems to be solved,
but the important role cytoskeleton plays in the cellular mechanotransduction is
widely recognised.

Besides that, cytoskeleton
network is a kind of spatial flexible structure that can be simulated by
mechanical model. Cytoskeleton is an extremely complex
biomolecular system in which molecular-scale components form a mesoscopic scale
structure together, and mesoscopic scale structure couples with each other to
form a continuous network. Some researchers use fractal dimension method to describe
this continuous and inhomogeneous structure and this method will be talked
later. From the viewpoint of bionics, the
cytoskeleton network has certain reference value for the study of spatial
structure. Scientists can find more effective method to support a structure
with limited materials by simulating the cytoskeleton network directly instead
of using computers to explore.

Researchers carried out a large number of experimental
studies to approach the truth of cytoskeleton gradually, and this article is
aimed at review and summarize some results. Due to the limited background and
level of the author, this essay only talks about some experiments and a simply
theoretical model.

 

Some Dynamic Characteristics about Cytoskeleton and Experiments

As the cytoskeleton system includes three filaments
and they connect to each other to form a complex network in a nanometre
dimension, so it is different for researchers manipulate the cell directly. Hence
many new experimental methods are used to discover the characteristics with
particular designed machines. Scientists usually mark the cytoskeleton network
with immunofluorescence and then scanned by laser scanner to analyse the
distribution and density of cytoskeleton.

Sonoporation is still a mystery to the cytoskeleton
network, which is how the cytoskeleton will behaviour like breakdown or
reorganization under a certain strength of Sonoporation. Except that, the
formation of pore by destroying the actin filaments or microtubules was studied
through various experiments, but is still undiscovered. 

Zeghimi and his team did not study the formation of
pore but compared the Sonoporation samples with control samples to explore the
some dynamic response on Sonoporation. They cultured human glioblastoma cells (U-87 MG) as the samples and exposed them under
ultrasound environment. In addition, these cells were marked with immunofluorescence
and then scanned by laser scanner. From the images shot by the laser scanner (Figure
2), the difference between filaments network is very obvious as the later
samples’ cytoskeleton reorganized the actin and microtubules. From further comparison,
this figure shows that the ‘combination of the ultrasound and microbubbles
increase the numbers of F-actin stress fibers’. (Zeghimi,
2014).
And after destroyed by ultrasound, the recovery of cytoskeleton is continuous and
the experimental samples (U-87 MG) has reorganized almost 92 percent protein
cytoskeleton after one hour.

Figure 2:Effect of sonoporation on ACTIN and TUBULIN cytoskeleton in U-87 MG.
White arrows show the actin and tubulin network in control cells, while
arrowhead designate the disorganization of cytoskeleton immediately after
sonoporation.

Considering the recovery
of sample cells, they used inhibitors to stop the recovery process and observed
the transmit ratio of SYTOX® Green was greatly decreased for 87 percentage. This
phenomenon can be viewed as an evidence on the transmit function of
cytoskeleton, but more researches are needed to prove this assumption.

Fractal dimension is a
theory which assume the detail parts change with the scale of measuring, and it
would be a great way to analyze complex space form. Qian and her team quantified
the MC3T3-E1 cells cytoskeleton with the fractal dimension analysis under
microgravity. Before their study, there are some research reported that the
cytoskeleton is going to alter under the spaceflight or weightless environment,
but the quantitative analysis had not been achieved. In this experiment, 3-D/2-D
Clinostats are used to simulate the weightless environment and operated in a
humidified incubator (5% CO 2 at 37 °C). Then using Leica TCS SP5 laser
scanning to record the cellular cytoskeleton after cells are cultured in the 3-D/2-D
Clinostats for 24 and 48 hours. NIH software ImageJ is used to quantify the
cytoskeleton images captured by Leica TCS SP5. In short, each complete cell
image see Figure 3 (a1) and (a2) is separated and translated into the 200 *
200 pixel gray value see Figure 3 (b1) and (b2). Through the automatic
threshold function of ImageJ (process> binary> make binary), the
grayscale image is converted to two valued image (black and white), which
contains only the contour information of the cytoskeleton as ROI see Figure 2
(c1) and c2. Then, based on box counting, ImageJ automatically determines the
size of the cytoskeleton see Figure 3 (d1) and (d2). The mean fractal
dimension (D) of more than 10 cells was calculated and analyzed statistically.

Fig. 3

They found the
microgravity environment do influence the distribution of F-Actin within 24
hours, but after 48 hours, the cytoskeleton can re-organize the structure and
recover to a common condition with the statistical data. However, the intensity
of ?-actin mRNA expression is different from the F-actin. It increased after 24
hours clinorotation to count the stress change, then the decrease shows the
adaption is almost complete after 48 hours. These findings indicate the microgravity
does limit damage to cytoskeleton with limit time and cells can recover from
the damage but will take relatively long time, but the results about cells
exposed to microgravity for enough has not been confirmed. (A.R.Qian, 2012)

The mechanics of normal
cells are different from cancer cells. Based on this principle, Shohreh and his
team intended to transform the cancer cells by restoring stiffness of abnormal
cells. In this experiment, they chose actin cytoskeleton as the target to
recover the elastic properties with the use of Lapatinib, a small molecule for
inhibiting EGFR over-activity. The over-activity of EGFR in the cells can lead
to the loss of mechanical properties like stiffness and adhesiveness because EGFR
is similar as the signal that influences the cells.

And the atomic force
microscopy was used to measure the mechanical properties with the formula:             

After treated by Lapatinib for 24 hours, the
mechanical properties were measured and analysed, so the following figure (Fig.
4) show the comparison between control cancer cells and Lapatinib about Young’s
Modules. And the Fig. 5 compare the actin filaments between two kinds of cells.

 

Figure 4.
Young’s modules difference

Figure 5. The actin filaments observed under the fluorescence microscope

These two figures
shows that Lapatinib stopped the signal pathway of EGFR, and recover the
mechanical properties obviously (Shohreh Azadi,
2016).
The mechanical properties including elasticity and adhesiveness influence cancer
cells metastasis, which will rise the generation speed of cancer cells. Now based
on the finding on Lapatinib, Lapatinib has been used in treating breast cancers
in the USA. But this results did not talk about the effect on Lapatinib
further, like: will this molecular will decrease the growth speed of breast
cancer cells or just make the cancer cell like the normal cells. This finding
may be a suggest that treat the cytoskeleton of cancer cells let them become or
partial become normal cells, as well as stop the relentless division and cancer
cells metastasis.

Besides these
experiment explored the characteristics on cytoskeleton, Deborah and his team tried
to improve the observation of cytoskeleton with the liner Hough Transform,
which is usually used to deal with the image information. This liner Hough
Transform is used in the software ImageJ. They tried different proper
constraints so that ‘only the actin filaments are discovered while spurious
points are ignored (those that happen to lie on a line but are not part of the
cytoskeleton structure)’ (Deborah Sturm,
2012).
In this experiment, they used mice cells to calculate, but the problem is
whether this constraints is useful for other kinds of cells.

Conclusion and discussion

As Alberts said in
his book: in many respects, we understand the structure of the universe better
than the workings of living cells. Although there are a great number of research
about cytoskeleton, the distance to the truth of it is still long. Scientist tried
various kinds of molecular and environments to stimulate the cytoskeleton, and
control variate method is commonly used.

In addition, there
are several challenges on cytoskeleton: 1. How to control the cytoskeleton
directly and clearly rather than using single element to affect the network? 2.
Setting up a quantized dynamic system about the response on cytoskeleton
network. 3. Building a digital model of cytoskeleton network, which can shows
the development and quantized values.