Cell Biology Lecture No. 11: Microtubules As Tracks For Transport
Wednesday February 13 , 2013
-By taking a squid axon (nerve) about 1mm in diameter and squish out all the cytoplasm, we can see all
kinds of structures like microtubules. Through adding chemicals like ATP, we can observe things moving
at different rates and in different directions along microtubules. This is because vesicle transport is bi-
directional and the motor proteins doing the transporting require ATP energy.
-The rate of axonal transport in vivo can be determined by radiolabeling and gel electrophoresis.
Radioactive amino acids injected into these cell bodies of neurons in experimental animals are
incorporated into newly synthesized proteins, which are then transported down the axon to the
synapse. These proteins can be identified after gel electrophoresis and autoradiography. The red, blue,
and purple dots represent groups of proteins that are transported down the axon at different constant
rates, either together or separately. Red moves most rapidly and purple least rapidly.
Kinesin As A Plus-End Directed Protein:
-There are many different classes of kinesin, the plus-end directed (ATP-dependent) motor protein of
microtubules (only a few exemplary cases show kinesin as a minus-end directed protein). Kinesin has 2
heavy chains (head, linker region and stalk) and 2 light chains (variable domain). The heavy chains
possess ATPase activity and microtubule-binding ability (quite conserved), while the light chains
recognize different types of cargo (thus the variability).
Structures Of Selected Kinesin Members:
-Three types of kinesin are important to consider: Type I, Type V and Type XIII. Kinesin-1 is more
conventional in nature to the usual description of the kinesin motor protein as it binds different cargo
and moves to the plus-end using ATP. Kinesin-3 has four heavy chains (no light chains) assembled in a
bipolar manner to interact with two antiparallel microtubules and also move toward the plus-end
(through a process known as sliding). Kinesin-13 family members have the motor domain in the middle
of their heavy chains (only head domains) and do not have motor activity, but they do
destabilize/disassemble microtubule ends (using ATP to remove dimers from ends).
-The cycle of anterograde movement starts with the leading head then binds ATP, which induces a
conformational change that causes the yellow linker region to swing forward and dock into its
associated head domain, thereby thrusting the trailing head forward. The new leading head now finds a
binding site 16 nm down the microtubule, to which it binds weakly. The leading head now releases ADP
and binds tightly to the microtubule, which induces the trailing head to hydrolyze ATP to ADP and P. P is i i released and the trailing head is converted into a weak binding state, and also releases the docked linker
Dynein As A Minus-End Directed Protein:
-Dynein is a minus-end directed motor protein and is involved in retrograde transport. The dynein heavy
chain heads have ATPase activity and the stalk domain. The linker region attaches the head to the stem
which in turn interacts with the dynactin hetero complex (that recognizes and binds to cargo). A number
of additional subunits associate with the stem region and link dynein to cargo through dynactin (links
dynein to cargo and regulates its activity). ATP hydrolysis can cause the shape of the linker region
(orientation of head relative to the stem) to change, driving movement of the microtubule-binding stalk.
Cilia & Flagella:
-Cilia and flagella are essentially two versions of the same thing, extracellular motion via stable
microtubules. Cilia (2-10 μm) are much shorter in length than flagella (10-2000 μm) and are represented
in greater abundance (since they sweep material across the tissue) than flagella (which propel cells in a
The Axoneme As The Underlying Structure Of Cilia & Flagella:
-The axoneme is a microtubule arrangement comprised of a 9 + 2 array (nine, linked, outer doublet
microtubules along with 2 inner