Biology Reference
In-Depth Information
shape of the micropattern, for example, forcing a polar axis on the cell, it is possible
to study how these parameters impact organelle organization, distribution, and dy-
namics inside the cell. To study the mitochondrial network, which is composed of
dynamic tubular organelles dependent on the microtubule cytoskeleton for its distri-
bution, it is important to be able to distinguish between distinct mitochondria. Here,
we present a practical method with which we spread the cells on large patterns cre-
ated with deep UV technique, which not only makes the cells uniform in size and
shape as well as immobile, and therefore easier to compare and analyze, but also ex-
pands the mitochondrial network and allows for an easier tracking of appropriately
labeled individual mitochondria.
INTRODUCTION
The mitochondria are often called the power plants of the cells, generating the ATP
the cell needs to carry out its many functions. They are also involved in many other
major physiological processes, such as reactive oxygen species biogenesis, calcium
homeostasis, and apoptosis. Generally thought of as bean shaped, mitochondria are
actually stretched out, tubular structures, which are highly dynamic (
Boldogh & Pon,
2007
). They constantly undergo fusion, fission, tubulation, and translocation
throughout the cell. Mitochondrial dynamics rely on either the actin or microtubule
cytoskeleton depending on species and cell type. Knowing the localization and dy-
namics of mitochondria in healthy and unhealthy cells is crucial for understanding
their functions and finding cures for the many mitochondria-based diseases that exist
(
Chan, 2006
).
Individual cultured cells can adopt many different shapes over time, which influ-
ence the positions and dynamics of organelles, making analysis difficult. Cells can
also be mobile, which makes viewing them over longer periods of time in a region of
interest complicated. Historically, mitochondria dynamics have often been studied in
neuronal axons (
Saxton & Hollenbeck, 2012
). This is due to the fact that axons
approximate regular 2D structures, where the mitochondria and their motions are
rectilinear and parallel to the axonal long axis, making dynamic analysis easily stan-
dardized. In other cultured cell types spread on a conventional substrate, where cells
adopt diverse shapes and are motile, the organization of mitochondria is not as
straightforward to study. To immobilize the cells and standardize cell sizes and
shapes, we applied micropatterning techniques using deep UV (
Azioune, Storch,
Bornens, Th
ยด
ry, & Piel, 2009
). This method enables us to image and analyze mito-
chondria and microtubule dynamics and organization in standardized cells. This
technique is straightforward to use in a typical biology lab, requires reasonably
simple equipments, and gives robust results. We describe below how the micro-
patterns are made and how the cells are attached and the mitochondria visualized.
This particular protocol is optimized for RPE1 cells but can be modified to study
mitochondria in other cell types as well.
