Cell bio paper

1. 2. 3. 4. 5. As stated in the introduction, what two roles had previously been identified for

XMAP215? (1 pt)

XMAP215 was previously shown to promote axon outgrowth by regulating microtubule

polymerization, as well as regulating the direction of growth cone trajectories.

What potential role for XMAP215 was being tested by the authors in this study? (1 pt)

The authors were testing the possibility that XMAP215 also regulates microtubule—F-

actin interactions.

The authors examine the effects of knockdown (KD) of XMAP215 on numerous growth

cone properties. How does knockdown of XMAP215 affect growth cone area, filopodia

length, growth cone pause number, the duration of growth cone pauses, length of

axons, and percentage of growth cones on ephrin stripes per explant, as shown in Figure

1? Be sure to comment on each one! (3 pts)

Knockdown of XMAP215 increases growth cone area and filopodia length. Knockdown

also increases the number of pauses and the duration of pauses, while decreasing axon

length. In addition, knockdown resulted in an increased percentages of growth cones on

ephrin stripes, suggesting knockdown results in a reduced ability to respond to repulsive

cues.

The authors next investigated the effect of knocking down XMAP215 on microtubule

morphology/shape. What type of microtubule morphology is observed at a higher

frequency upon XMAP215 knockdown as shown in Figure 2A? Are the microtubules

more or less dynamic than controls, as shown in 2B? (1 pt)

Knockdown of XMAP215 resulted in an increase in microtubules that exhibited a looped

morphology, as well as microtubules that were more dynamic than controls.

Next, the authors examine the ability of XMAP215 to regulate microtubule penetration

in the growth cone. They observe and increase in the number of exploring microtubules

overall, but they are also interested in determining if these exploring microtubules are

present in filopodia. How does the percentage of exploring microtubules in filopodia

compare in knockdown (KD) samples relative to the control in Fig. 3E? (1 pt)

The authors observe a reduced percentage of exploring microtubules in filopodia in KD

samples relative to controls.

6. 7. 8. 9. In Figure 4, the authors examine alignment of F-actin with microtubules in the growth

cone. What effect does knockdown (KD) of XMAP215 have on the alignment of F-actin

and microtubules? What is the effect does overexpression (OE) of XMAP215 have on

alignment? (2 pts)

XMAP215 knockdown results in a significant decrease in the percentage of microtubules

aligned to F-actin, while overexpression increases the percentage of aligned

microtubules and F-actin.

To investigate which domains are needed for microtubule-F-actin alignment, Slater et

al., generate a variety of deletion mutants to test. Which region of XMAP215 is needed

for the newly discovered functions of this protein based on the data in Figure 6? (1 pt)

The TOG1-5 region is most important for the newly discovered functions of XMAP215

because expression of this region is enough to rescue the defects associated with

XMAP215 knockdown.

What assay/experiment was used to test the ability of XMAP215 to bind directly to F-

actin in Figure 7? What did the results of this experiment show? (2 pts)

Co-sedimentation assays were used to test if XMAP215 binds directly to F-actin.

Experiments demonstrated that XMAP215 does interact with F-actin.

What is one question that still remains about how XMAP215 functions to regulate the

cytoskeleton (look at the Discussion for ideas)? (1 pt)

Lots of possibilities here. A few possible answers:

Can XMAP215 bind microtubules and F-actin at the same time? Are additional proteins

needed for XMAP215 to bind to F-actin? How does XMAP215 lead to changes in growth

cone size and filopodial length? What feedback mechanisms may be functioning among

XMAP215, microtubules and Factin?