Nerve Cell

1. Microtubules Intermediate Filaments Actin Microfilaments How about prokaryote? Helicobacter Nerve Cell Vibrio Erythrocyte Egg cell Stella Caulobactercrescentus Lipocyte

2. The Bacterial Cytoskeleton：An Intermediate Filament-Like Function in Cell Shape Nora Ausmees, Jeffrey R. Kuhn, and Christine Jacobs-Wagner

3. Before… • Townsend (in 1980), Trachtenberg and Gliad (in 2001 and 2003) found there are some wall-less bacteria, such as spiral-shaped Spiroplasma, an unconventional cytoskeleton is involved in motility and cell shape deformation.

4. Before… • Mutations in several genes invovled in peptidoglycan metabolism have been show to ALTER the cell morphology of various bacteria. And NO intracellular filaments were found. • ASSUMPTION: Cell-walled bacteria are DEVOID of a cytoskeleton that supports cell shape.

5. Before… • Studies on Escherichia coli and Bacillus subtillis have shown a protein with weak sequence similarity to actin, is essential for determining the rod shape of these bacteria. • Recent evidence suggests a coupling between the biosynthesis of the peptidoglycan and the bacteria actin-like cytoskeleton.

6. It still remains unclear… • How more complex and often asymmetric shapes can be achieved in bacteria? • Here, we will introduce you about the author’s experiment and it’s result.

7. How a IF-like protein, crescentin, is required for determining the shape of Caulobacter crescentus.

8. Caulobacter crescentus • Caulobacter crescentus is a Gram-negative, oligotrophic bacterium widely distributed in fresh water lakes and streams. It is an important model for studying the regulation of the cell cycle and cellular differentiation. • It always be observed in two shapes, vibrioid and helical, which is depending on the length of the cell.

9. Identification of Crescentin, an Essential Determinant of the Curved and Helical Shapes of C. crescentus

10. Identification • Performed a visual screen of a library of random transposon (Tn5) insertion mutants. • There are two independent Tn5 insertion, one is 228 bp downstream and the other one is 15 bp upstream of the start condon of the ORF CC3699. • Finally, identified two mutant clones with a straight-rod morphology.

11. Identification

12. Identification • Thus, crescentin is required for both the vibrioid and helical shapes of C.crescentus.

13. Crescentin Forms a Filamentous Structure that Colocalizes with the Inner Cell Curvature

14. Form • Most of its sequence has a distinct 7-residue repetitive pattern predicted to form coiled-coils. Coiled-coils are the main structural elements of many fibrous proteins in eukaryotes. • This, together with the involvement of crescentin in shape determination, suggested crescentin may support shape by forming an intracellular filament.

15. Colocalize

16. Colocalize

17. Colocalize

18. The Subcellular Localization of the Crescentin Filamentous Structure Is Important for Causing Cell Curvature

19. Localization • The asymmetric subcellular localization of crescentin structure not only demonstrates at the molecular level the existence of a bacterium, but it is also likely to pertain to the cytoskeletal function of crescentin in cell curvature.

20. Localization

21. Localization • The helical crescentin structure applies its helical geometry to the cells, presumably via a direct or indirect interaction with the cytoplasmic membrane.

22. Crescentin Is a Bacterial Cytoskeletal Protein Similar to Eukaryotic Intermediate Filament Proteins

23. Sequence Similarity • Crescentin shares sequence similarity with IF proteins • The sequence of crescentin shares 25% identity and 40% similarity (over 254 residues) with that of cytokeratin 19. • And 24% identity and 40% similarity (over 397 residues) with that of nuclear lamin A.

24. Sequence Similarity Stutter’s position in the C-terminal coiled-coil segment is highly conserved among vertebrate and invertebrate IF proteins. Human IF proteins

25. In Vitro Ability Similarity 1. The most distinctive property of IF proteins is their in vitro ability to form filaments raging from 8 to 15 nm in diameter without a requirement for divalent cations, nucleotides, or other exogenous factors. Filaments are obtained by simply dialyzing the protein against physiological or low-ionic strength buffers at a neutral pH. 2. A purified polyhistidine-tagged version of crescentin spontaneously self-assembled into filaments with a width of about 10 nm after removal of 6M guanidinium by dialysis against a 50mM Tris-HCl buffer at pH 7.0. The formation of His-CreS filaments was severely impeded in buffers at pH 8.4 and above. This behavior is commonfor IF proteins. Crescentin has the defining biochemical property of IF-related proteins to self-assemble into filaments in vitro without the need for energy or cofactors.

26. Why Is C.crescentus Curved?

27. Why Is C.crescentus Curved? • “Friendly” laboratory conditions • Motile • No defects of cell division or chromosome segregation——grow normally • Sustained viability • In nature • A dilute aquatic environment, cell dispersal • Exploration of new environment for nutrients relies on motility. • Propelled by a helical flagellum

28. How Represented Is the IF-like Cytoskeletal Function across Kingdoms?

29. Across Kingdoms Various arrangements of coiled-coil regions may give rise to similar IF-like biochemical properties. yeast H.pylori 4 uncharacterized coiled-coil proteins from Helicobacter pylori.

30. Across Kingdoms • In plants, no IF proteins has been described thus far. • A recent genomic study aimed to investigate coiled-coil proteins from Arabidopsis did not find proteins with the same domain organization as animal IF proteins. • A family of uncharacterized proteins with extended coiled-coils. Some members of which may fulfill an IF-like function.

32. Conclusion • Crescentin in Caulobacter crescentus • Function——form vibrioid and helical shapes • Crescentin is similar to intermediate filaments • Other IF-like cytoskeletal function across kingdoms

33. THANK YOU！