Each is a __of __subunits held together by weak, __ bonds
What does this allow?
Contains microtubules, microfilaments, and intermediate filaments that together form an elaborate interactive network
polymer
protein subunits
noncovalent
This allows rapid assembly and disassembly, which is dependent on complex cellular regulation
Microtubules
long, hollow, unbranched tubes composed of subunits of the protein tubulin
microfilaments
Microfilaments: solid, thinner structures, often organized into a branching network and composed of the protein actin
intermediate filaments
Intermediate filaments: tough, ropelike fibers composed of a variety of related proteins
True or False:
Both prokaryotes and eukaryotes have cytoskeletal or cytoskeletal-like structures
true
Functions 1 and 2
Dynamic scaffold providing structural support that can determine the shape of the cell and resist forces that tend to deform it
Internal framework responsible for positioning organelles within cell
Functions 3 and 4 and 5
Network of tracts for movement of materials and organelles
Force-generating apparatus that moves cells from ne place to another
Single-celled organisms crawl or propel themselves through their aqueous environment with help of locomotor organlelles (cilia and flagella)
Essential component of the cell’s division machinery
Make up mitotic spindle
Experiments
Concept of __ first derived from tissues viewed with __in 1950s-60s
Couldn’t show dynamics of it-->__ and __
__proteins are synthesized with cell fused with __
cytoskeleton
EM
fluorescence microscope and live-cell imaging
Fluorescently labeled
GFP
In an alternate approach, the protein subunits of cytoskeletal structures are __labeled __by covalent linkage to a small __
Labeled subunits then microinjected into living cell where incorporated into __, like a microtubule
Its then followed
fluorescently
in vitro
fluorescent dye
polymeric form of protein
If particularly small amounts of fluorescently labeled proteins are injected, the cytoskeletal filaments are no longer what?, but produce __. This is called __.
uniformly labeled
speckles that serve as fixed markers to follow dynamic changes in length or orientation of the filament
fluorescence speckle microscopy
Fluorescence microscopy can be used to reveal __within cell of what?
_-used because they can ____
location
a protein present in low concentrations
Antibodies
distinguish between closely related variants of a protein
Protein __by injecting the labeled __into a living cell, which may also reveal the function of the target protein because attachment of the __generally does what?
Location can also be determined how?
localized
antibody x2
destroys the ability of the protein to perform in its normal activity
by adding the fluorescent antibody to fixed cells or tissue sections
In vitro motility assays make it possible to do what?
These make it possible to detect the activity of an individual protein molecule acting as a molecular motor in “real time
In some earlier assays, microtubules were attached to a __. Then, micron-sized beads containing attached __were placed onto __by __, which are shone through the objective lens of the microscope, producing a __ near the point of focus
Called __
glass coverslip
motor proteins
microtubules
laser beams
weak attractive point
optical tweezers
When ATP is present, the movement of the __along a microtubule can be followed by a __
__can be used to trap a single bead and determine its forces
FLAW: __
beads
video camera
Focused laser beams
This is indirect because it monitors motility of the beads, not protein molecules
Direct visualization of movement by combining __with __
__from other areas in the field of view do not obscure the information that emanates from the individual protein molecules whose activities are being monitored
fluorescence microscopy
specialized imaging techniques
Fluorescent light rays
What does it allow
comparison of properties of different motor proteins
measuring of the properties of individual motors under different experimental conditions
asking how a specific mutation of a motor protein affects its motility properties
Fluorescently labeling __and fluorescently labeled __watch interaction of the two types of labeled proteins as they take place within the cell’s cytoplasm under various physiological conditions
microtubule polymers
kinesin molecules
Although light-microscopic fluorescence techniques can allow researchers to follow the movements of individual protein molecules, they cannot __
Consequently, these optical techniques do not tell us much about the __
resolve the molecules themselves
molecular mechanisms by which these proteins actually operate
On the other hand, __ and __ are well-suited for revealing the structure of the molecules but can only provide snapshots of the molecule at a single stage in its operation
electron microscopic and X-ray crystallographic techniques
New technique allowing high resolution and observation in real time; uses __
Used to measure the __
The development of techniques for working with single molecules has coincided with the creation of a new field of mechanical engineering called __
AFM
mechanical properties of cytoskeletal elements
nanotechnology
Goal and uses of nanotechnology
development of tiny “nanomachines” capable of performing specific activities in a submicroscopic world
Hopefully, one day, such machines can carry out functions in human body
Dynamic structures are continually undergoing some type of __, whether their old parts are being exchanged with new ones or they are being entirely broken down and rebuilt.
Thus, even though a __ or __appears __from one moment to the next, its molecular components are in a __
rebuilding
microtubule or actin filament
unchanging
state of flux
True or False:
Changes occur constantly during the cell cycle. Monitoring this requires good technique
True
Fluorescence recovery after photobleaching (FRAP)
How to carry out FRAP
cell is either injected with tubulin coupled to a fluorescent dye or induced to express GFP-tubulin.
Using a microscope with a __that can be focused on a small region of the cell, the fluorescence of the __in that region is __by the __
Measure the recovery of the signal into the __
laser
tubulin
leached
laser beam
bleached zone
What can recovery be from?
dynamics of the microtubules turning over in that bleached zone, growth of new microtubules into the bleached zone, or movement of mcirotubules through the bleached zone
