RESUMEN

The effects of changes in the loading rate during the forced dissociation of single bonds have been studied for a wide variety of interactions. Less is known on the loading rate dependent behaviour of more complex systems that consist of multiple bonds. Here we focus on viral nanoparticles, in particular the protein shell (capsid) that protects the viral genome. As model systems we use the well-studied capsids of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) and of the bacteriophages φ29 and HK97. By applying an atomic force microscopy (AFM) nanoindentation approach we study the loading rate dependency of their mechanical properties. Our AFM results show very diverse behaviour for the different systems. In particular, we find that not only the breaking force, but also the spring constant of some capsids depend on the loading rate. We describe and compare the measured data with simulation results from the literature. The unexpected complex loading rate dependencies that we report present a challenge for the current theoretical considerations aimed at understanding the molecular level interactions of highly ordered protein assemblies.

Asunto(s)

Bacteriófagos/fisiología , Bacteriófagos/ultraestructura , Bromovirus/fisiología , Bromovirus/ultraestructura , Cápside/fisiología , Cápside/ultraestructura , Fenómenos Mecánicos , Fenómenos Biomecánicos , Microscopía de Fuerza Atómica , Nanopartículas/ultraestructura

RESUMEN

The elastic properties of micrometer-sized hollow colloidal particles obtained by emulsion templating are probed by nanoindentation measurements in which point forces are applied to solvent-filled particles supported on a flat substrate. We show that the shells respond linearly up to forces of 7-21 nN, where the indentation becomes of the order of the shell thickness (20-40 nm). In the linear region, the particle deformation is reversible. The measured Young's modulus (approximately 200 MPa) is comparable to values for stiff rubbers or soft polymers. At larger applied force, we observe a crossover into a nonlinear regime, where the shells assume a buckled shape. Here, the force increases approximately as the square root of the indentation, in agreement with the theory of elasticity of thin shells. We also observe permanent deformation of the shells after probing them repetitively beyond the linear regime. Finally, the measured elastic properties of the shells nicely explain their spontaneous buckling in solution and due to drying.

RESUMEN

The main functions of viral capsids are to protect, transport and deliver their genome. The mechanical properties of capsids are supposed to be adapted to these tasks. Bacteriophage capsids also need to withstand the high pressures the DNA is exerting onto it as a result of the DNA packaging and its consequent confinement within the capsid. It is proposed that this pressure helps driving the genome into the host, but other mechanisms also seem to play an important role in ejection. DNA packaging and ejection strategies are obviously dependent on the mechanical properties of the capsid. This review focuses on the mechanical properties of viral capsids in general and the elucidation of the biophysical aspects of genome packaging mechanisms and genome delivery processes of double-stranded DNA bacteriophages in particular.

Asunto(s)

Bacteriófagos/fisiología , Cápside/química , Empaquetamiento del ADN , ADN Viral/metabolismo , Genoma Viral/fisiología , Bacteriófagos/genética , ADN Viral/genética

RESUMEN

At least in mammals, we have some understanding of how caspases facilitate mitochondria-mediated cell death, but the biochemical mechanisms by which other factors promote or inhibit programmed cell death are not understood. Moreover, most of these factors are only studied after treating cells with a death stimulus. A growing body of new evidence suggests that cell death regulators also have 'day jobs' in healthy cells. Even caspases, mitochondrial fission proteins and pro-death Bcl-2 family proteins appear to have normal cellular functions that promote cell survival. Here, we review some of the supporting evidence and stretch beyond the evidence to seek an understanding of the remaining questions.

Asunto(s)

Apoptosis/fisiología , Supervivencia Celular/fisiología , Mitocondrias/fisiología , Animales , Bacterias/citología , Humanos , Saccharomyces cerevisiae/citología

RESUMEN

The elastic properties of capsids of the cowpea chlorotic mottle virus have been examined at pH 4.8 by nanoindentation measurements with an atomic force microscope. Studies have been carried out on WT capsids, both empty and containing the RNA genome, and on full capsids of a salt-stable mutant and empty capsids of the subE mutant. Full capsids resisted indentation more than empty capsids, but all of the capsids were highly elastic. There was an initial reversible linear regime that persisted up to indentations varying between 20% and 30% of the diameter and applied forces of 0.6-1.0 nN; it was followed by a steep drop in force that is associated with irreversible deformation. A single point mutation in the capsid protein increased the capsid stiffness. The experiments are compared with calculations by finite element analysis of the deformation of a homogeneous elastic thick shell. These calculations capture the features of the reversible indentation region and allow Young's moduli and relative strengths to be estimated for the empty capsids.

Asunto(s)

Bromovirus/ultraestructura , Proteínas de la Cápside/genética , Cápside/ultraestructura , ARN Viral/ultraestructura , Bromovirus/genética , Elasticidad , Genoma Viral/genética , Concentración de Iones de Hidrógeno , Microscopía de Fuerza Atómica , Mutación Puntual

RESUMEN

The shell of bacteriophages protects the viral DNA during host-to-host transfer and serves as a high-pressure container storing energy for DNA injection into a host bacterium. Here, we probe the mechanical properties of nanometer-sized bacteriophage phi 29 shells by applying point forces. We show that empty shells withstand nanonewton forces while being indented up to 30% of their height. The elastic response varies across the surface, reflecting the arrangement of shell proteins. The measured Young's modulus (approximately 1.8 GPa) is comparable with that of hard plastic. We also observe fatigue and breakage of capsids after probing them repetitively. These results illustrate the mechanoprotection that viral shells provide and also suggest design principles for nanotechnology.

Asunto(s)

Bacteriófagos/química , Cápside/química , Elasticidad , Estrés Mecánico

RESUMEN

Centrin/Cdc31p is a Ca2+-binding protein related to calmodulin found in the MTOC of diverse organisms. In yeast, Cdc31p localizes to the SPB where it interacts with Kar1p and is required for SPB duplication. Recent findings suggest that centrin also functions elsewhere in the cell. To dissect the functions of Cdc31p, we generated cdc31 mutations chosen only for temperature sensitivity, but otherwise unbiased as to phenotype. Three phenotypes of the cdc31 mutants, temperature sensitivity, G2/M arrest, and cell lysis, were not well correlated, indicating that the mutations may differentially affect Cdc31p's interactions with other proteins. Alleles near the C-terminal region exhibited high G2/M arrest and genetic interactions with kar1-Delta17, suggesting that this region modulates an SPB-related function. Alleles causing high lysis and reduced Kic1p kinase activity mapped to the middle of the gene, suggesting disruption of a KIC1-like function and defects in activating Kic1p. A third region conferred temperature sensitivity without affecting cell lysis or G2/M arrest, suggesting that it defines a third function. Mutations in the C-terminal region were also defective for interaction with Kic1p. Mapping the alleles onto a predicted structure of Cdc31p, we have identified surfaces likely to be important for interacting with both Kar1p and Kic1p.

Asunto(s)

Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona , Mapeo Cromosómico , Proteínas de Saccharomyces cerevisiae , Alelos , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calmodulina/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , División Celular , Proteínas Fúngicas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Mutación , Proteínas Nucleares/genética , Fenotipo , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , Estructura Terciaria de Proteína , Programas Informáticos , Huso Acromático/metabolismo , Supresión Genética , Temperatura , Técnicas del Sistema de Dos Híbridos

RESUMEN

The earliest known step in yeast spindle pole body (SPB) duplication requires Cdc31p and Kar1p, two physically interacting SPB components, and Dsk2p and Rad23p, a pair of ubiquitin-like proteins. Components of the PKC1 pathway were found to interact with these SPB duplication genes in two independent genetic screens. Initially, SLG1 and PKC1 were obtained as high-copy suppressors of dsk2Delta rad23Delta and a mutation in MPK1 was synthetically lethal with kar1-Delta17. Subsequently, we demonstrated extensive genetic interactions between the PKC1 pathway and the SPB duplication mutants that affect Cdc31p function. The genetic interactions are unlikely to be related to the cell-wall integrity function of the PKC1 pathway because the SPB mutants did not exhibit cell-wall defects. Overexpression of multiple PKC1 pathway components suppressed the G2/M arrest of the SPB duplication mutants and mutations in MPK1 exacerbated the cell cycle arrest of kar1-Delta17, suggesting a role for the PKC1 pathway in SPB duplication. We also found that mutations in SPC110, which encodes a major SPB component, showed genetic interactions with both CDC31 and the PKC1 pathway. In support of the model that the PKC1 pathway regulates SPB duplication, one of the phosphorylated forms of Spc110p was absent in pkc1 and mpk1Delta mutants.

Asunto(s)

Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/genética , Proteínas Quinasas Activadas por Mitógenos , Proteína Quinasa C , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Huso Acromático/genética , Western Blotting , Proteínas de Unión a Calmodulina , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/metabolismo , Fase G2 , Regulación Fúngica de la Expresión Génica , Prueba de Complementación Genética , Proteínas de la Membrana/genética , Mitosis , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Fosforilación , Saccharomyces cerevisiae/genética , Temperatura , Factores de Tiempo , Ubiquitinas/genética

RESUMEN

Saccharomyces cerevisiae cells decide to divide during G1. If nutrients are abundant, cells pass through START and coordinately undergo DNA replication, bud emergence, and spindle pole body duplication. Phenotypic analysis of the slg1delta mutant revealed that this mutation uncouples post-START events. At the nonpermissive temperature, slg1delta cells that have undergone bud emergence but not DNA replication or SPB duplication accumulate. Furthermore, while wild-type cells arrest in GO when starved, the slg1delta mutant fails to arrest at this point; instead, cells with small buds accumulate. The slg1delta mutation displayed genetic interactions with cdc34, which encodes a regulator of exit from G1. This is consistent with a role of SLG1 in G1 regulation. Epitope-tagged Slg1p cofractionated with the plasma membrane, suggesting that Slglp may function by integrating external cues and relaying them to the interior of the cell. We propose that SLG1 plays a regulatory role in bud emergence or stationary phase.

Asunto(s)

Fase G1/fisiología , Glicoproteínas de Membrana/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Secuencia de Bases , Ciclo Celular/genética , Ciclo Celular/fisiología , Cartilla de ADN/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Fase G1/genética , Genes Fúngicos , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Mutación , Fase de Descanso del Ciclo Celular/genética , Fase de Descanso del Ciclo Celular/fisiología , Saccharomyces cerevisiae/genética , Temperatura

RESUMEN

We have isolated two human ubiquitin-like (UbL) proteins that bind to a short peptide within the ATPase domain of the Hsp70-like Stch protein. Chap1 is a duplicated homologue of the yeast Dsk2 gene that is required for transit through the G2/M phase of the cell cycle and expression of the human full-length cDNA restored viability and suppressed the G2/M arrest phenotype of dsk2Delta rad23Delta Saccharomyces cerevisiae mutants. Chap2 is a homologue for Xenopus scythe which is an essential component of reaper-induced apoptosis in egg extracts. While the N-terminal UbL domains were not essential for Stch binding, Chap1/Dsk2 contains a Sti1-like repeat sequence that is required for binding to Stch and is also conserved in the Hsp70 binding proteins, Hip and p60/Sti1/Hop. These findings extend the association between Hsp70 members and genes encoding UbL sequences and suggest a broader role for the Hsp70-like ATPase family in regulating cell cycle and cell death events.

Asunto(s)

Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Saccharomyces cerevisiae , Ubiquitinas/genética , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Relacionadas con la Autofagia , Proteínas de Ciclo Celular/química , Proteínas Fúngicas/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Alineación de Secuencia , Ubiquitinas/química , Ubiquitinas/metabolismo

RESUMEN

KAR1 is required for duplication of the Saccharomyces cerevisiae microtubule organizing center, the spindle pole body (SPB) (Rose, M.D., and G.R. Fink, 1987. Cell. 48:1047-1060). Suppressors of a kar1 allele defective for SPB duplication were isolated in two genes, CDC31 and DSK2 (Vallen, E.A., W.H., M. Winey, and M.D. Rose. 1994. Genetics. 137:407-422). To elucidate the role of DSK2 in SPB duplication, we cloned the gene and found it encodes a novel ubiquitin-like protein containing an NH2 terminus 36% identical to ubiquitin. The only other known yeast ubiquitin-like protein is encoded by the nucleotide excision repair gene RAD23 (Watkins, J.F.,P. Sung, L. Prakash, and S. Prakash. 1993. Mol. Cell. Bio. 13:7757-7765). Unlike ubiquitin, the NH2-terminal domain of Dsk2p is not cleaved from the protein, indicating that Dsk2p is not conjugated to other proteins. Although the DSK2-1 mutation alters a conserved residue in the Dsk2p ubiquitin-like domain, we detect no differences in Dsk2p or Cdc31p stability. Therefore, DSK2 does not act by interfering with ubiquitin-dependent protein degradation of these proteins. Although DSK2 is not essential, a strain deleted for both DSK2 and RAD23 is temperature sensitive for growth due to a block in SPB duplication. In addition, overexpression of DSK2 is toxic, and the DSK2-1 allele causes a block in SPB duplication. Therefore, DSK2 dosage is critical for SPB duplication. We determined that CDC31 gene function is downstream of DSK2 and KAR1. Dsk2p is a nuclear-enriched protein, and we propose that Dsk2p assists in Cdc31 assembly into the new SPB.

Asunto(s)

Centrosoma/fisiología , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Ubiquitinas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Unión al Calcio/análisis , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/genética , Centrosoma/ultraestructura , Clonación Molecular , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/análisis , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/fisiología , Datos de Secuencia Molecular , Peso Molecular , Mutación , Proteínas Nucleares/genética , Procesamiento Proteico-Postraduccional , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Ubiquitinas/biosíntesis , Ubiquitinas/química , Ubiquitinas/fisiología