QUIMIOTAXIS.

Presentación del tema: "QUIMIOTAXIS."— Transcripción de la presentación:

1 QUIMIOTAXIS

2 Cómo se mueve E. coli : corridas + tumbos = exploración al azar
CCW: CORRIDA CW: TUMBO E. coli swims by rotating it flagellar filaments, which operate much like the propellers on an ocean liner. Each flagellum has a rotary motor at its base that can turn the propeller in either direction: counter-clockwise (CCW) rotation allows the individual flagella to form a coherent bundle that pushes the cell forward; clockwise (CW) rotation of any one of the motors disrupts the bundle. The individual filaments work against one another, causing the cell to tumble about randomly. Tumbles serve to reorient the cell's swimming direction when it begins another run. Tumbling episodes occur with constant probability, i.e., they are Poisson-distributed. Some runs are brief, others are long. Overall, the cell's motions approximate a random walk, comprised of forward movements punctuated by abrupt, random directional changes.

3 Una exploración al azar sesgada permite seguir un gradiente
amino ácidos azúcares Detección del gradiente: temporal (memoria ~3") alta sensibilidad (~0.1% ∆C/C) Amplio rango (5-6 logs) Integración de estímulos E. coli is attracted to some sugars and amino acids and repelled by a variety of noxious compounds such as ethanol. In chemoeffector gradients, the cells move toward attractants and away from repellents by modulating their tumbling episodes. Whenever, the cell happens to travel in a favorable direction, it senses that conditions are improving and uses that information to reduce the probability of the next tumble. Because of its small size, E. coli is constantly knocked off course by Brownian motion, so it can only make new progress toward favorable chemical environments in a biased random walk fashion. Movements in the favorable direction last a bit longer than movements in other directions. E. coli senses spatial chemical gradients by using a clever memory mechanism to make temporal concentration comparisons while swimming about. The cell constantly compares current conditions to those averaged over the past 3-4 seconds in its travels. In this short comparison window, the cell can sense concentration differences as small as one part in a thousand and its retains high sensitivity over nearly a million-fold concentration range. These are extraordinary signaling feats for a supposedly "simple" cell and genetic studies have played a central role in revealing the molecular mechanisms of this remarkable behavior.

4 Placas de “swimming” serina Agar blando Atrayente metabolizable Gradiente generado por el consumo Movimiento coordinado siguiendo el gradiente aspartato

5 Mutantes en genes de quimiotaxis
Ciega a serina (Tsr-) tryptone, 32°C, 8 hr. Anillo de serina Anillo de aspartato salvaje (Tar+ Tsr+) Ciega a aspartato (Tar-) Here is a simple assay for chemotactic behavior. The plate contains a rich medium solidified with a low concentration of agar. The cells can swim through water-filled tunnels in the agar matrix. As they grow, they consume nutrients, primarily serine and aspartic acid, that are attractant compounds. Their metabolic activity creates attractant gradients, which they will follow, if they can. Thus, wild-type colonies exhibit two discrete rings that contain chemotactic pioneers: their outer ring represents a serine-seeking group (because this is the first amino acid consumed); the inner ring represents an aspartate-seeking group . These cells are NOT genetically different from those in the first ring; they just never experienced a serine gradient because they happened to grow up in the wrong part of the colony. Mutants lacking the chemoreceptors (detectors) for serine or aspartate make colonies that lack one of these rings. Mutants that can swim, but cannot carry out any chemotactic responses, make small colonies that expand slowly through random swimming motions. Mutants that cannot swim make even smaller colonies because they cannot leave the inoculation site. No móvil (Fla-, Mot-) No quimiotáctica (Che-)

6 Placas de swimming: método óptimo para aislamiento de mutantes
pCS12 (wt Tsr) pCS12 R388A pCS12 R388A*

7 Método en capilares

8 Ensayo del tapón de agarosa

9 Sistema de señalización relativamente simple
Notable conservación en las proteínas de señalización Capacidad de detectar gradientes químicos con exquisita sensibilidad Capacidad de detectar gradiente en un amplio rango de concentraciones Integración y amplificación de señales

10 W A Y P Los niveles de CheY-P determinan cierta frecuencia de cambios de dirección

11 W A Y Los atrayentes inhiben la kinasa, REDUCIENDO la frecuencia de cambios de dirección

12 EXCITACION Cambio en la actividad de la kinasa (y por lo tanto en la frecuencia de cambios) en respuesta a un gradiente de atrayente o repelente ADAPTACION Regreso a la frecuencia de cambios inicial (antes del estímulo). Permite a la bacteria la detección de cambios de concentración en un rango de 5 órdenes de magnitud. Mediada por la metilación reversible de los receptores. Provee a la célula de una especie de memoria.

13 MCPs: Methyl-accepting Chemotaxis Proteins
Tsr: serina Tar: aspartato y maltosa (MBP) Tap: dipéptidos Trg: ribosa (RBP) y galactosa (GalBP) Aer: O2 (estado redox?) Aer

14 Vía de transducción de señales involucrada

15 Los quimiorreceptores se agrupan en los polos de la célula
Maddock & Shapiro, 1993 Zhang, et al., 2007 Sourjik & Berg, 2000 The basis of receptor-receptor communication is thought to be physical clustering of the chemoreceptors in patches at the cell pole(s). This might enable receptors of different types to exchange sensory information and collaborate in the control of CheA output signals. The receptor array might behave like a very large allosteric enzyme complex.

16 Dominio citoplasmático de Tsr
Estructura de MCPs

17 Existen interacciones laterales entre distintos receptores?
W A Tar Tsr

18 Tris-(2-maleimidoethyl) amine
S366C (Tsr) es un buen indicador de la formación de trímeros (a través de crosslinking con TMEA) Tsr S366C TMEA Tris-(2-maleimidoethyl) amine O-O distance: 10.4 Å

19 Ensayo de crosslinking para detectar trímeros de dímeros in vivo

20 Modelo para la formación del complejo de receptores
Trímeros de dímeros: tienen un papel esencial en la señalización?

21 Green fluorescent protein (GFP)
Before 1999, the functional form of MCP-family receptors was thought to be a homodimer, as shown. (NB: the structure of the HAMP domain, which transmits conformational changes induced by ligand-binding in the periplasmid domain to the cytoplasmic output domain, remains a mystery.) However, in the Kim group’s crystal, three dimers of the Tsr signaling domain were associated in a trimer of dimers. To determine whether the trimer of dimers had physiological significance, we created and characterized a set of Tsr mutants with amino acid replacements at individual trimer contact residues. The phenotypes of those mutants led us to propose that receptors operate in collaborative signaling teams, presumably based on trimers of dimers. Piston & Kremers, 2007

22 Características espectrales de las proteínas fluorescentes
spectral overlap between CFP and YFP CFP emission YFP excitation Fluorescence resonance energy transfer (FRET) is a technique that measures the separation of two fluorescently labelled proteins (and hence their interaction) in cells. It relies on distance-dependent energy transfer from an excited donor fluorophore to an acceptor fluorophore. Because FRET-based measurements are quantitative and non-invasive, FRET is particularly useful for observing transient protein interactions involved in signal transduction. For in-vivo FRET, proteins of interest can be expressed as fusions to cyan fluorescent protein (CFP, donor) and yellow fluorescent protein (YFP, acceptor), and energy transfer can be measured by exciting CFP fluorescence and monitoring fluorescence intensity in two spectral channels corresponding to CFP and YFP emissions. Interactions between fusion proteins result in energy transfer from CFP to YFP, thereby quenching CFP fluorescence and inducing YFP fluorescence.

23 Ensayo FRET in vivo para la actividad kinasa de CheA
figures from Sourjik, 2004 Sourjik & Berg, 2002

24 Detección de FRET CheZ-CFP/CheY-YFP
figure from Sourjik et al., 2007 Sourjik & Berg, 2002

25 CheY-YFP + CheZ-CFP Estas dos proteínas interactúan cuando CheY está fosforilada, por lo que el cambio en la fluorescencia relativa es una medida casi directa de la actividad de la kinasa CheA

26 Organización de los genes