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PhysicalFactorsAffectsBudFormationPattern
inWildTypeYeastModel(Saccharomyces
cerevisiae)
Article·August2013
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Current Research Journal of Biological Sciences
ISSN:
© Maxwell Scientific Organization, 2013
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Accepted:
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Physical Factors Affects Bud Formation Pattern in Wild Type Yeast Model
(Saccharomyces cerevisiae)
.M. Ogundele1O, 2O.A. Alao, 3O.O. Idris, 5O.J. Olajide, 1A.O. Adekeye, 1L.A. Enye,
1
O.O. Ogedengbe, 1P.A. Adeniyi, 4J.O. Sanya, 1D.A. Adekomi and 3B.A. Oso
1
Department of Anatomy, College of Health Sciences,
2
Department of Physical Sciences, College of Sciences,
3
Department of Microbiology, College of Sciences,
4
Department of Physiology, College of Health Sciences, Afe Babalola University, Ado-Ekiti, Nigeria
5
Department of Anatomy, College of Health Sciences, University of Ilorin, Ilorin, Nigeria
Abstract: The pattern of cell division in the yeast models suggests that the process of cell division is not
spontaneous but the direction of such cell divisions is predetermined by several physical factors. The study is aimed
at investigating the mathematical relationship involved in cell division (in the budding yeast) and how it can be
affected by certain physical factors (light and gravity). The hypothesis was further tested using live yeast models
that were sub-cultured in potatoe dextrose agar and kept at room temperature on a microscope stage. The
illumination technique was adjusted to reduce the intensity of the incident light. The image was recorded on the
computer interface to determine time dependence and effects of light and gravity in determining the direction of
division and cell movement in bud formation in the S. Cerevisiae. The angle of budding φN+2 at event N+2, was
observed to be dependent on φN and φN+1 as ∆φ = φN-φN+1, where ∆φ = φN+2 and are positive for successive
buds.
Keywords: Bud angle, cell division, culture, budding, microtubule, microscope, yeast
The specific aim is to predict probable angle
formation pattern between parent yeast cells and
successive buds during the modeled cell division
putting the MTOC positions and spindle angle relative
to the ring dimensions in perspective. Also to determine
the direction of bud formation in relation to physical
factors such as temperature, water, light and gravity and
how these prediction models could be extrapolated to
determine the role of physical factors in determining
migration of tumor cells in vivo. In this study, the scar
is taken as the base of the parent organism and the point
of origin from which a perpendicular line will be drawn
to determine angle of deviation from the origin [see
hypothesis section].
INTRODUCTION
The budding yeasts are tiny single celled fungi
whose mechanism of cell cycle control is remarkably
similar to those of the human species and are thus used
in cell cycle and cell division experiments (Cáp et al.,
2012). The family of cell division proteins in the yeast
models are conserved, so also are the centrioles and the
spindle formation mechanism. The centrioles are built
on microtubule proteins which have been conserved for
over a billion years from the yeast to humans in
phylogenetic analysis (McIntosh et al., 2010; Duncker
et al., 2009). Budding yeast is typical as it has features
and hypothetical shapes close to that of the mammalian
cell and divides in a manner close to that of the
mammalian cells (Segal and Bloom, 2001).
Budding is the predominant mode of vegetative
reproduction in yeast and multilateral budding is a
typical feature of budding yeast (Calahan et al., 2011).
The bud formation is a function of cell size of the
parent yeast and it will normally coincide with the
replication time and onset of DNA synthesis. This is
followed by localized weakening of cell wall due to
uneven distribution of MT pull force towards one side
of the ring (Markus et al., 2012).
HYPOTHESIS
Figure 1 in this hypothesis, the scar is taken as the
point origin from which divergence will be measured.
Using image analysis system from different
experiments at different times for differently cultured
budding yeast, a predominant pattern was obtained for
all unfixed yeast colonies such that the sum of angle i
and d equals 800 ; thus indicating the deviation around
the ring of the bud at its origin from the parent yeast.
Corresponding Author: O.M. Ogundele, Department of Anatomy, College of Health Sci., Afe Babalola University, Ado-Ekiti,
Nigeria
1
Curr. Res. J. Biol. Sci.,
Fig 1: Schematic illustration of the prevalent pattern of a yeast colony with origin N and buds N+1,N+2………K. The relative
bud angles φ and the angles i and d between a parent yeast cell and a subsequent bud. βi and βd represents respective
distances of the spindle from angles i and d, while MTOCN represents the relative position of the centriole in each event
(Image courtesy Dr. Olalekan Ogundele, Cell biology and Histology Research Lab, Afe Babalola University, Nigeria)
The wider angle between successive yeast opposite
the direction of budding is represented as φ for event N,
N+1 and N+2. Extrapolating the data from pictures of
colonies the difference in φN and φN+1 gives the
approximate size of angle φN+2, thus:
∆φ = φN-φN+1
βi≠βd
but βi is greater than βd thus generating a tension
towards angle i and tilt of the bud towards angle i. This
implies that ∆φ which in this scenario is φN+2 is a
function of MTOC and β for events N, N+1,
N+2……….K, where K is the equilibrium point where
∆φ is negative and a bud leaves the colony to start a
new colony. When cell moves, they move in the i angle
as a result of tension generated on βi and is dependent
on MTOC N, N+1, N+2…….K.
(1a)
This pattern predicts the approximate position of
the third successive bud from the relative angle of the
first and second buds such that:
φ = φN + 3
(2)
(1b)
Generalizing Equation 1: The events for successive
buds φN and φN+1 can be expressed as a probability of
change in bud angle φ. Considering the fact that the
yeast models exists in artificial environment under the
influence of physical factors, it is therefore, to express
the change in angle at successive buds (∆φ) as a
function of deviation of the angle (φ) at events N, N+1,
N+2…..K, where K is the equilibrium state where a
negative value is obtained for ∆φ.
The exception for this rule is that if ∆φ = φN-φN+1
gives a negative value then there is a change in the
direction of the next bud which will detach from the
colony to start a new colony with scar as point of origin
or the base. The implication of this is that the number of
consecutive buds by a parent yeast cell is predetermined
by the angle of its first bud and the negative value
before its detachment from the parent colony.
It is imperative to note the Eq. (1) is dependent of
spindle location and the relative distance of the spindle
to the angles i and d which is represented by βi and βd
respectively. The position and spindle angle is
determined by the relative position of the centrioles
(MTOC). In our study the spindles and the MTOC are
closer to the angle d such that MTOCN and MTOC N+1
are on a straight-line thus:
Equation 2: the probability that deviation at φN and
φN+1 will occur as non ordered event can be expressed
as φN, φN+1 = P(N,N+1), while the frequency of event N
and N+1 will be expressed as:
Equation 3: φN, φN+1= P (N,N+1) + P(N+1,N).
Thus: φN, φN= 2 P (N,N+1) [3b]
2 [3a]
Curr. Res. J. Biol. Sci.,
Since both events will occur till point K, the likely hood
of occurrence of event φN is therefore:
φN = ∑
(4)
φN≠φN+1
(5)
This further supports the ideology that the angle φ
will change in each successive bud till a negative is
obtained at equilibrium “K”.
MATERIALS AND METHODS
Yeast culture: Potatoe dextrose agar (culture medium)
was used and prepared as thus; 0.5 g K2HPO4, 0.2 g
MgSO4.7H20, 0.2 g NaCl, 0.2gCaCl2.6H20, 10 g potatoe
dextrose agar and 0.4g of extract (Spadaro et al., 2010).
The medium was dispensed and sterilized. The vial
containing the yeast specimen (WT) was carefully
opened and stripped on the culture media in a glass
petri dish.
Secondary culture: A thin film of the yeast culture
medium was placed on a separate petri dish and a
secondary culture of single yeast cell was prepared with
sucrose being added as 1% v/v in the secondary culture
to facilitate division of the yeast to form buds.
The yeast cells were stained with Coomasie
brilliant blue G250 (1mg/mL stock solution) which was
made to a final concentration of 2.5 µg/mL. The
secondary culture was grown for 30 more min post
staining. Before microscopy the cells were washed in 9
Phosphate Buffer Saline (PBS) and were finally resuspended in 9 Phosphate Buffer Saline (PBS)
(Swayne et al., 2009).
Fig 2: Brightfield imaging of live yeast (WT) in culture
(rendered in grayscale); P represents the parent bud,
D: daughter bud and arrow head indicates the scar for
the previous bud and the present bud, C: cytoplasmic
materials. In Figure 2A and B, note the elongation of
some yeast buds in response to light, this is believed to
be negative phototaxis that will affect the direction
and degree of budding as seen in A1 and A2 in 2
different set ups. A single colony is represented in the
dashed white line coloured area. Starting from the
origin (blue arrow) represented by the scar on the
parent bud P at event n, n+1,n+2 and n+3. Where
subsequent origins are indicated by sites of bud
(yellow arrows) for events n to n+1, n+1 to n+2 and
n+2 to n+3 (Magnification X1,000).
Treatment: Different secondary culture plates were
then subjected to mild temperature ranging from 35-45
°C, Ultraviolet (UV) radiation and gravity (by
positioning the culture plates in a side position rather
than in an upright position.
Image acquisition and analysis: The Olympus bright
field research microscope was used, a video (digital)
camera attachment MV 550 was placed in one of the
ocular of the research microscope and was connected to
a computer system for live cell imaging and real time
recording of the budding process in the cultured and
stained yeast cells. The acquired images were processed
on Open Office Draw (JAVA) for measurements and
label lings.
and 3). Also gravity affected the buds of specific yeast
cells in Fig. 2A and B such that the buds were
elongated and larger in a symmetrical manner.
Comparing these findings to the control yeast, the
budding deviated from the normal expected budding
direction observed in the control yeast cells (Fig. 4A
and B). This shows that the direction of cell division
can be predetermined by certain physical factors; aside
the intrinsic molecular control mechanisms factors like
temperature, light and gravity will probably affect the
final direction of budding and the size and shape of the
RESULTS
DISCUSSION AND CONCLUSION
The budding yeast showed negative phototaxis by
turning away from the source of light (visible) (Fig. 2
3 Curr. Res. J. Biol. Sci.,
Fig. 3: Deviation in the budding pattern in yeast cells exposed to visible light at 360C. Note the wide deviation in angle i and d
compared to the control yeast cells (Magnification X1, 000. 3A is the schematic illustration of the budding patter, 3B is
the actual bud formation patter. φN is the budding angle at event n, φN2 is the budding angle at event N+2. Observe the
sharp deviation in the budding angle at event N2
Fig. 4: Normal budding yeast not subjected to any physical parameter. Figure 4A is the schematic illustration while the yellow
area in Fig. 4B indicates the sample budding yeast used for the schematic drawing
bud as seen in the buds which responded to gravity by
producing elongated buds in the direction of gravity. Of
importance is the fact that amidst several budding cells
very few produced such elongated buds. The reason
behind this is not clear but it is suspected to be strain
difference with respect to sensitivity to physical
parameters. The proposed role of MTOC position and
inter spindle distance cannot be mathematically proven
in this current study but opens opportunity for future
research where these measurements are obtainable at
higher magnifications in electron microscopy.
The budding process is not random and cellular
geometry is important in determining the bud site.
Several studies have shown that genetic and molecular
mechanisms underlie the selection of budding site on a
yeast cell (Magwene et al., 2011). The possibility of
having the axial budding pattern depends on the
combination of a and α haploid cells while a/α will
normally give a bi-polar budding pattern where the
daughter cell buds away from the mother cell and the
mother cell buds towards or away from the daughter
cell (Vopálenská et al., 2005; Segal et al., 2000).
Mitotic spindle must align along the symmetry of cell
polarity, taking its origin from the parent scar (Segal
and Bloom, 2001); this mechanism depends on
localization of centrioles (MTOC) and a precise
programme of cues originating from the cell cortex
(Yang et al., 1997; Segal et al., 2000). The mechanism
of spindle pole polarity is primed by the symmetrical
alignment of the MTOC on an asymmetrical plane in
relation to the ring and the scar (Markus et al., 2012).
This study uses yeast models to predict probable
direction of cell movement after division or budding S.
Cerevisiae and the possible influence of physical
factors like temperature, light, nutrients and gravity in
determining the pattern of such division and movement.
In a yeast colony, the scar of a primary bud is the origin
4 Curr. Res. J. Biol. Sci.,
of such organism from its parent organism while the
ring of the bud from such organism is the head or
proximal region of the organism. The pattern of
budding in S. Cerevisiae is not in a symmetrical manner
(not straight), thus the bud is tilted towards one side
(Markus et al., 2012). The budding follows the
hypothetical centriole and spindle pattern (centriole is a
microtubule organization centre; MTOC) which are
tilted towards one side of the ring (the ring being the
connection between the parent and the bud governed by
a family of proteins CDC42 and Septin); this will in
turn cause the bud to tilt towards one side (Roemer
et al., 1996). The location of the MTOC in the parent
yeast is always above the food vacuole, thus giving the
spindle convex angulations. This phenomenon is
different for the fission yeast whose cytoskeleton forms
rings that detaches on a straight-line from either ends
(Valtz and Herskowitz, 1996; Segal et al., 2000). A
similar protein controls cell division and migration in
the human tumor cells and the yeast. The CDC42 of the
S. Cerevisia is a homologue of the tumor CDC42.
While the CDC42 functions in late state excocytosis it
has also been found to be a major contributor and
regulator of cell polarity (Roemer et al., 1996). Mutants
in which the gene has been silenced were observed to
be unable to form buds, in other mutants where, there is
over expression of the gene, it was found not to have
any deleterious effect but affected the randomness of
the positioning of the bud. The Cdc42 in tumor cells
has been observed to be a critical regulator of tumor
cell-endothelial cell interaction via its control on β1intergrins (Reymond et al., 2012). The Cdc42 have also
been implicated in the in vivo cancer spreading just as
its homologue in the yeast CDC42 controls direction of
bud formation and spread in the yeast colony. This gene
represents a noteworthy overlap point in yeast and
cancer biology, it also shows a complex array of
phylogenetic relationship between migrating tumor
cells and the budding yeast. The homologues both
interact with a complex array of proteins to regulate
microtubule, cytoskeleton and membrane dynamics.
The studies of Reymond et al. (2012) shows that
depletion of Cdc42 reduced cancer cell spreading,
likewise the deletion of the yeast CDC42 stopped bud
formation in the yeast models (Shinjo et al., 1990).
where by cell divided and migrates uncontrollably. The
word ‘uncontrollably” needs to be re-evaluated as a
possible control and determinant of such migration in
the body involves arrays of physical factors and
nutrients or metabolites specific for various forms of
cancer cells. The primary significance of this negative
phototaxis remains unclear but it is a clear indication
that cells without photo receptors posses machineries
for interpreting light as chemical signals.
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