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Structure of Biological Macromolecules and Macromolecular
Assemblies; Membrane Structure and Function; Electron Microscopy and Image Processing
Our
research interests are the structural study of biological
assemblies by application of biophysical and biochemical
methods. More specifically, we apply techniques of
high resolution electron microscopy and digital image
processing to study the structures of biological macromolecules,
macromolecular assemblies, and whole organelles. Currently
we are studying the structure and function of mitochondria
using state of the art microscopic techniques, principally
Electron Tomography. Electron Tomography is a technique
which calculates the three-dimensional structure from
a series of electron micrographs of cells or cellular
components tilted over a range of angles. Our study
of mitochondria has led, along with the work of several
other research groups, to a new paradigm of mitochondrial
structure in which the inner membrane of mitochondria
is divided into two components. The inner boundary
membrane is the component that lies along the outer
membrane separated from it by approximately 3-7 nanometers.
At numerous sites we find 30 nm diameter tubular extensions
of the inner membrane projecting into the matrix toward
the center of the mitochondrion forming cristae, the
second component of the inner membrane. Numerous tubular
cristae often merge forming large disklike cristae.
These results have raised a number of questions about
mitochondrial structure and function that we are currently
addressing in collaboration with groups at SDSU and
other research institution (See Frey and Mannella
2000 and MitoMovies)
(1) Is the mitochondrial
inner membrane compartmentalized? Although the
inner boundary membrane and the cristae membrane are
one continuous surface, they are connected by discrete
tubular crista junctions that are only 30nm in diameter.
This suggests that the functions of the inner membrane
may be compartmentalized by controlling the distribution
of inner membrane proteins (See Frey et al.
2002).
(2) Do cristae
grow from the addition of membrane proteins and lipids
at tubular crista junctions? To answer this question
we have studied the loss of crista in Neurospora
mitochondria inhibition ofTom20, a critical component
in protein import. Cristae membrane and crista junctions
are lost at the same rate and precede loss of outer
membrane and inner boundary membrane. (see Perkins
et al. 2001).
(3) What are the
changes in mitochondria structure during apoptosis?
Mitochondria play a key role in initiating and/or
regulating the apoptosis program with the release
of cytochrome c and other proteins into the
cytosol. We are using correlated confocal light microscopy
and electron microscopy/tomography to study the structural
changes in mitochondrial membrane conformation during
apoptosis using different cell culture models. Our
goal is to determine whether cytochrome c is
released through specific pores in the outer membrane
or by rupture of the outer membrane following swelling
of the matrix. In this context we are also studying
the possible role of the mitochondrial permeability
transition and "remodeling" of the inner
mitochondrial membrane in cytochrome c release
4) What effects
do high concentrations of reactive oxygen species
(ROS) have on the structure of mitochondria? Ischemia/reperfusion
injury during a heart attack is believed to result
an increase in reactive oxygen species that stimulates
release of cytochrome c from mitochondria
initiating apoptosis and/or necrosis. We are studying
the effects of increasing ROS on cytochrome c release
and changes in mitochondrial structure by correlated
light and electron microscopy/tomography of mitochondria.
5)
What are the physical chemical factors that control
mitochondrial structure? In collaboration with
colleagues in the Department of Math and Department
of Physics we are refining the thermodynamic model
developed by Renken et al. (2002) to explain
the stability of mitochondrial membrane conformations
observed by electron tomography.
Representative Publications
Frey, T.G.
and Perkins, G.A. (2006) “Electron Tomography
of Intracellular Organelles” Ann. Rev.
Biophys. Biomolec. Struct. (in press).
A. Ponnuswamy, Nulton,J.,
Mahaffy, J.M., Salamon, P., Frey, T.G.,
and Baljon, A.R.C. (2005) “Modeling tubular
shapes in the inner mitochondrial membrane”
Physical Biology 2, 73-79.
Frey, T.G.,
Renken, C.S., and Perkins, G.A. (2002) "Insight
into mitochondrial structure and function from electron
tomography." Biochim. Biophys. Acta
1555, 196-203.
Renken, C.W., Siragusa,
G., Perkins, G., Washington, L., Nulton, J., Salamon,
P., and Frey, T.G. (2002) "A thermodynamic
model describing the nature of the crista junction,
a structural motif in the mitochondrion." J.
Struct. Biol. 138, 137-144.
Perkins, G.A., Renken,
C.W., van der Kleij, I.J., Ellisman, M.H., Neupert,
W., and Frey, T.G. "Electron tomography of
mitochondria after the arrest of protein import
aided by TOM19." Europ. J. Cell Biol.
80, 139-150 (2001).
Frey, T.G.
and Mannella, C.A. "The internal structure
of mitochondria." Trends in Biochemical
Sciences 25 319-324 (2000).
Ph.D. students: Christian
Renken
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