Areas of Interest
Research in this laboratory is focused on molecular mechanisms
of intracellular transport and organization of microtubule
cytoskeleton. The model system that is being used is
melanophores, pigment cells of lower vertebrates. The only
function of these large cells is synchronous transport of
thousands of membrane-bounded organelles, pigment granules,
which rapidly move to the cell center to form a tight aggregate
or redisperse uniformly throughout the cytoplasm. During
aggregation, pigment granules move along microtubules by means
of cytoplasmic dynein. Pigment dispersion involves initial rapid
microtubule-dependent transport to the periphery by Kinesin II
and subsequent slow diffusion-like movement along the randomly
arranged actin filaments. Transport is regulated by Protein
Kinase A (PKA) signaling cascade. Thus, melanophores provide a
unique model system for the studies of the role of cytoskeleton
in intracellular transport, mechanisms of switching between the
two major transport systems, and regulation of activity of motor
molecules by signal transduction mechanisms.
Two recent findings define the directions of current
research. First, we have shown that in microsurgically produced
cytoplasmic fragments of melanophores lacking the centrosome the
radial array of microtubules rapidly forms and becomes
positioned to the center. Thus, membrane organelles that are
normally dragged by motors to the centrosome region may
themselves play an active role in organization and maintenance
of radial microtubules. Digital fluorescence microscopy,
photobleaching , photoactivation and microinjection of
motor-specific probes are being used to test the mechanisms of
self-organization and self-centering of the radial microtubule
array in the fragments. Second, we have demonstrated that during
dispersion the pigment granules that initially move along
microtubules switch tracks and continue motion along randomly
arranged actin filaments. Thus, each pigment granule bears a
member of each of the families of motor molecules: cytoplasmic
dynein and a kinesin-like motors (specific for microtubules) and
a myosin motor (specific for actin filaments). A combination of
biochemical and molecular approaches are being used to test the
hypothesis that the motor molecules interact and that regulation
is achieved through phosphorylation of common subunits.
Lab Rotation Projects
1. Identification of Protein Kinase A adapter proteins (AKAPs).
Our recent work indicates that PKA is bound to pigment granules
and that this binding is mediated by adapter proteins know as
AKAPs. A combination of biochemical, molecular, and
mass-spectrometry approaches will be used to identify AKAPs that
tie PKA to pigment granule surface and determine the role of PKA
compartmentalization in pigment transport.
2. Mechanism of regulation of Kinesin II. Our preliminary
data strongly suggest that the activity of kinesin II during
dispersion is stimulated by PKA-dependent phosphrylation.
Mass-spectrometry, site-directed mutagenesis, and microscopy
approaches will be used to identify phosphorylation sites and
determine the importance of Kinesin II regulation for pigment
dispersion.
3. The role of actin dynamics in pigment dispersion. During
dispersion, pigment granules move along the actin filaments by
means of myosin Va. Based on our data we hypothesize that this
actin-dependent transport involves continuous growth of actin
filaments. Live imaging approaches will be used to test this
hypothesis and determine whether actin assembly is coupled to
myosin V activity.
Selected Publications
Malikov, V., A.Kashina, and V.Rodionov. 2004. Cytoplasmic
dynein nucleates microtubules to organize them into the radial
array. Mol. Biol. Cell. 15:2742-2749.
Cytrinbaum, E., V.Rodionov, and A.Mogilner. 2004.
Computational model of dynein-dependent self-organization of
microtubule asters. J. Cell Sci. 117:1381-1397.
Burakov, A.V., E.S.Nadezhdina, B.Slepchenko, and V.I.Rodionov.
2003. Centrosome positioning in interphase cells. J. Cell
Biol.162:963-969.
V.Rodionov, J.Yi, A.Kashina, A.Oladipo, and S.P.Gross. 2003.
Switching between microtubule- and actin-based transport systems
in melanophores is controlled by cAMP levels. Curr. Biol.
13:1-20.
Vorobjev, I.V., V.P.Malikov, and V.I.Rodionov. 2001.
Self-organization of a radial microtubule array by dynein-dependent
nucleation of microtubules. Proc. Natl. Acad. Sci. USA
98:1060-1065. |
