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Faculty
 Vladimir
Rodionov
Assistant Professor of Cell Biology
rodionov@nso.uchc.edu
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. |
