microtubule based movement, final question

[ValWood]

I went through all (most?) the experiements about movement of proteins along mictotubule in tea2 delete.

So, in PMID:12034771 behrans Tea1p is transported from the nucleus to the cell ends on the tips of microtubules http//:www.jcb.org/ cgi/content/full/jcb.200112027/DC1 In all cases, tea1- GFP dots first appeared in the vicinity of the nucleus. In this region, the direction of the tea1GFP dots changed fre- quently, but once the dots moved out of the vicinity of the nucleus, they moved processively towards the cell ends. We conclude that tea1p is loaded on microtubules in the vicinity of the nucleus, that changes in the direction of movement can occur within the loading region, and that once tea1p has moved beyond that region, it generally moves processively toward the cell ends. n the vicinity of the nucleus, changes in the direction of tea1YFP movement were usually confined to bright regions of the microtubules which have been reported to contain overlapping ends of antiparallel microtubule bundles (Drummond and Cross, 2000; Tran et al., 2001). Tea1YFP dots were found to be present on the tips of microtubules and the dots remained colocalized with the tips of polymerizing microtubules while moving from the nucleus to the cell ends (Fig. 1 C) 3.6 +/-1.um/min similar to the rate of polymerization of GFP-labeled microtubules The frequency with which tea1GFP dots were delivered to cell ends was also measured and found to be 0.86 +/- 0.63 min-1 end-1

• The tea2p kinesin is not required for tea1p movement near the nucleus

Then In cells lacking the tea2p kinesin, there is a major reduction of tea1p at the cell ends, indicating that tea2p is required for tea1p localization at the cell ends (Browning et al., 2000) In >95% of the tea2delta cells, tea1GFP was absent from both cell ends or was present at one end but at significantly re- duced levels (Fig. 2 A) To investigate whether the tea2p ki- nesin is required for the movement of tea1p, we determined the speed of tea1GFP dot movement in tea2 delta cells. A typical time course is shown in Fig. 2 B http//:www.jcb.org/cgi/content/full/jcb.200112027/DC1 We conclude that in the absence of the tea2p kinesin, tea1p associates normally with the tips of polymerizing microtubules and can move with a rate similar to that observed in wild-type cells, but that this movement is confined primarily to the central region of the cell. (a) The transport of the morphogenetic factor tea1p in fission yeast occurs at the tips of polymerizing microtubules, moving processively from the vicinity of the nucleus towards the ends of the cell; (b) The tea2p kinesin is not required for tea1p movement on the tips of polymerizing microtubules, but in the absence of tea2p moving tea1p is confined to the vicinity of the nucleus; (c) A COOH-terminal coiled-coil re- gion of tea1p is required for the efficient retention of the protein at the cortex of the cell ends; and (d) The localization of tea1p at the cell ends is not required for the termination of microtubular growth at the cell ends, but is required for the retention of the morphogenetic factors tea2p, tip1p, and pom1p at the cell ends, and for proper cell morphogenesis.

• So this does not preclude the possibility that another kinesis is operating in the nuclear region, and tea2 only kicks in further on?

Then in PMID:12894167 Browning H, Hackney DD, Nurse P Tea2 is loaded onto microtubules in the middle of the cell, in close proximity to the nucleus, and then travels using its intrinsic motor activity primarily at the tips of polymerizing microtubules. The microtubule-associated protein Mal3, an EB1 homologue, is required for loading and/or processivity of Tea2 and this function can be substituted by human EB1. In addition, the cell-end marker Tea1 is required to anchor Tea2 to cell ends. Movement of Tea1 and the CLIP170 homologue Tip1 to cell ends is abolished in Tea2 rigor (ATPase) mutants. We propose that microtubule-based transport from the vicinity of the nucleus to cell ends can be precisely regulated, with Mal3 required for loading/processivity, Tea2 for movement and Tea1 for cell-end anchoring.

This paper goes on to say

a) Potential cargoes for Tea2 include Tea1 and the CLIP170 homologue Tip1 (refs 4, 9). Tea1 is a cell-end marker required for cells to maintain their linear growth pat- tern; it is transported by microtubules to the cell ends where it accu- mulates10. Although Tea1 can still move, it is distributed all along the microtubules in tea2∆ cells instead of concentrated at microtubule tips4,11 (also see Fig. 3b) B. In addition, Tea1–GFP and Tip1–YFP were unable to move in tea2-rigor mutants and colocalized with the rigor mutant proteins (data not shown). Thus, Tea2 may affect microtubule dynamics and cell shape by ensuring the efficient delivery of these and other cargo proteins to microtubule tips and cell ends. C Although the microtubule cytoskeleton is defective in tea2∆ and tea2 rigor mutants, the inability of Tea1 and Tip1 to localize to the microtubule tips and cell ends was not a result of these defects. Microtubule polymerization rates in these strains were similar to wild- type cells: ……. In addition, some microtubules grew to the cell end: 71% for tea2∆ . Those microtubules that reached the cell ends were localized there for less time in mutant cells, compared with wild-type cells: …..These results suggest Tea2 prevents microtubule catastrophe as the polymer grows to the cell end and once it reaches the cell end, perhaps functioning through Tip1 and other cargo proteins.

However- I don’t see tha C is a reason for B? kinesin-dependent movement along a microtuble doesn't necessarity require microtubule polymerisation also, does it?

Based on the a above, I would say that the rate of movement in the central region (where it still occurs) is the same as WT, but that movement along microtubules was not "normal" because it is confined to the region close to the nucleus. This seems very similar to the tea1-tip1 experiments. I'll insert figures.....

[ValWood]

also this figure, busch fig 1

and fig 4

[ValWood]

So, I'm not sure that we should say that tea2 has normal transport/movement of tea1

My problem is this term: http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0099112 Biological Process Definition The transport of a protein driven by polymerization of a microtubule to which it is attached.

because I don't think we know that the movement is "based on polymerization". Tea1 could be moved by another kinesin (at normal rate) closer to the nucleus, but require tea2 for normal transport to the cell tip?

(this could also have been a misintepretation of mine during GO term requesting)

[ValWood]

@jvhayles

Can discuss by Skype later... I might be missing something.

[ValWood]

Jacky from older ticket In PMID1203477, they show that the rate of tea1GFP movement on microtubules is the same in wild type and tea2 Delta strains suggesting tea2 is not moving tea1GFP Fig2B, and they also show that tea1GFP co-localises with the growing microtubule ends. I think the cell tip localisation is indirect and the accumulation of tea1 in the vicinity of the nucleus is indirect because tea2 affects the length of the microtubules. Does this help I'll also have a look at PMID 11018050

So this might be what you are saying. If we thing the transport role of tea2 might be indirect we won't make any GO annotation for this part.

I'm worried that we are conflating the microtubule polymerization with the transport along the microtubule. We can discuss the possible scenarios...