Nerve Cell

Erythrocyte

Egg cell

Lipocyte

Helicobacter

Vibrio

Stella

Caulobacter crescentus

Microtubules

Intermediate

Filaments

Actin

Microfilaments

How about

prokaryote?

The Bacterial The Bacterial CytoskeletonCytoskeleton ：： An An Intermediate Filament-Intermediate Filament-Like Function in Cell Like Function in Cell ShapeShape

Nora Ausmees, Jeffrey R. Kuhn, and Christine Jacobs-Wagner

Before…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.

Before…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.

Before…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.

It still remains It still remains unclear…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.

HowHow a IF-like protein, a IF-like protein, crescentincrescentin, is required for , is required for determiningdetermining the shape of the shape of Caulobacter crescentusCaulobacter crescentus. .

Caulobacter crescentusCaulobacter crescentusCaulobacter 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.

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

IdentificationIdentificationPerformed 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.

IdentificationIdentification

IdentificationIdentification

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

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

FormFormMost 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.

ColocalizeColocalize

ColocalizeColocalize

ColocalizeColocalize

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

LocalizationLocalizationThe 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.

LocalizationLocalization

LocalizationLocalization

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

Crescentin Is a Bacterial Crescentin Is a Bacterial Cytoskeletal Protein Cytoskeletal Protein Similar to Eukaryotic Similar to Eukaryotic Intermediate Filament Intermediate Filament ProteinsProteins

Sequence SimilaritySequence SimilarityCrescentin 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.

Sequence SimilaritySequence Similarity

Human IF proteins

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

In Vitro Ability In Vitro Ability SimilaritySimilarity

IF proteins

crescentin

Self-assemble + +

Method to obtain proteins

dialysis dialysis

observe + +

Prevent from

forming

pH 8.4 and

above

pH 8.4 and

above

Crescentin has the defining biochemical property of IF-related proteins to self-assemble into filaments in vitro without the need for energy or cofactors.

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 common for IF proteins.

Why Is Why Is C.crescentusC.crescentus Curved?Curved?

Why Is Why Is C.crescentusC.crescentus Curved?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

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

Across KingdomsAcross Kingdoms

yeast

H.pylori

Various arrangements of coiled-coil regions may give rise to similar IF-like biochemical properties.

4 uncharacterized coiled-coil proteins from Helicobacter pylori.

Across KingdomsAcross KingdomsIn 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.

ConclusionConclusionCrescentin in Caulobacter

crescentusFunction——form vibrioid and

helical shapesCrescentin is similar to

intermediate filamentsOther IF-like cytoskeletal function

across kingdoms

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