Ajit P. Joglekar

The kinetochore encodes a mechanical switch to disrupt spindle assembly checkpoint signalling

The spindle assembly checkpoint (SAC) is a unique signalling mechanism that responds to the state of attachment of the kinetochore to spindle microtubules. SAC signalling is activated by unattached kinetochores, and it is silenced after these kinetochores form end-on microtubule attachments. Although the biochemical cascade of SAC signalling is well understood, how kinetochore–microtubule attachment disrupts it remained unknown. Here we show that, in budding yeast, end-on microtubule attachment to the kinetochore physically separates the Mps1 kinase, which probably binds to the calponin homology domain of Ndc80, from the kinetochore substrate of Mps1, Spc105 (KNL1 orthologue). This attachment-mediated separation disrupts the phosphorylation of Spc105, and enables SAC silencing. Additionally, the Dam1 complex may act as a barrier that shields Spc105 from Mps1. Together these data suggest that the protein architecture of the kinetochore encodes a mechanical switch. End-on microtubule attachment to the kinetochore turns this switch off to silence the SAC.

The Budding Yeast Point Centromere Associates with Two Cse4 Molecules during Mitosis

The centromere is defined by the incorporation of the centromere-specific histone H3 variant centromere protein A (CENP-A). Like histone H3, CENP-A can form CENP-A-H4 heterotetramers in vitro. However, the in vivo conformation of CENP-A chromatin has been proposed by different studies as hemisomes, canonical, or heterotypic nucleosomes. A clear understanding of the in vivo architecture of CENP-A chromatin is important, because it influences the molecular mechanisms of the assembly and maintenance of the centromere and its function in kinetochore nucleation. A key determinant of this architecture is the number of CENP-A molecules bound to the centromere. Accurate measurement of this number can limit possible centromere architectures. The genetically defined point centromere in the budding yeast Saccharomyces cerevisiae provides a unique opportunity to define this number accurately, as this 120-bp-long centromere can at the most form one nucleosome or hemisome. Using novel live-cell fluorescence microscopy assays, we demonstrate that the budding yeast centromere recruits two Cse4 (ScCENP-A) molecules. These molecules are deposited during S phase and they remain stably bound through late anaphase. Our studies suggest that the budding yeast centromere incorporates a Cse4-H4 tetramer.

Assembling the Protein Architecture of the Budding Yeast Kinetochore-Microtubule Attachment using FRET

BACKGROUND The kinetochore is a multiprotein machine that couples chromosome movement to microtubule (MT) polymerization and depolymerization. It uses numerous copies of at least three MT-binding proteins to generate bidirectional movement. The nanoscale organization of these proteins within the kinetochore plays an important role in shaping the mechanisms that drive persistent, bidirectional movement of the kinetochore. RESULTS We used fluorescence resonance energy transfer (FRET) between genetically encoded fluorescent proteins fused to kinetochore subunits to reconstruct the nanoscale organization of the budding yeast kinetochore. We performed >60 FRET and high-resolution colocalization measurements involving the essential MT-binding kinetochore components: Ndc80, Dam1, Spc105, and Stu2. These measurements reveal that neighboring Ndc80 complexes within the kinetochore are narrowly distributed along the length of the MT. Dam1 complex molecules are concentrated near the MT-binding domains of Ndc80. Stu2 localizes in high abundance within a narrowly defined territory within the kinetochore centered ∼20 nm on the centromeric side of the Dam1 complex. CONCLUSIONS Our data show that the MT attachment site of the budding yeast kinetochore is well organized. Ndc80, Dam1, and Stu2 are all narrowly distributed about their average positions along the kinetochore-MT axis. The relative organization of these components, their narrow distributions, and their known MT-binding properties together elucidate how their combined actions generate persistent, bidirectional kinetochore movement coupled to MT polymerization and depolymerization.

Nanochannels fabricated by high-intensity femtosecond laser pulses on dielectric surfaces

Direct scanning electron microscopy examination reveals a complex structure of narrow, micron-deep, internal nanochannels within shallow, nanoscale, external craters fabricated on glass and sapphire surfaces by single high-intensity femtosecond laser pulses, with nearly the same intensity thresholds for both features. Formation of the channels is accompanied by extensive expulsion of molten material produced via surface spallation and phase explosion mechanisms, and redeposited around the corresponding external craters. Potential mechanisms underlying fabrication of the unexpectedly deep channels in dielectrics are considered.

Plasticity and Epigenetic Inheritance of Centromere-specific Histone H3 (CENP-A)-containing Nucleosome Positioning in the Fission Yeast

Background: CENP-A nucleosomes support kinetochore assembly. Results: Cnp1/CENP-A occupancy, indicated by silencing of the reporter gene, varies among genetically homogenous cells. Conclusion: Centromeric chromatin organization is flexible and inherited epigenetically. Significance: How CENP-A nucleosomes are inherited is potentially important for assembling one functional centromere per chromosome and may serve as an experimental system to elucidate the general mechanisms for epigenetic inheritance. Nucleosomes containing the specific histone H3 variant CENP-A mark the centromere locus on each chromatin and initiate kinetochore assembly. For the common type of regional centromeres, little is known in molecular detail of centromeric chromatin organization, its propagation through cell division, and how distinct organization patterns may facilitate kinetochore assembly. Here, we show that in the fission yeast S. pombe, a relatively small number of CENP-A/Cnp1 nucleosomes are found within the centromeric core and that their positioning relative to underlying DNA varies among genetically homogenous cells. Consistent with the flexible positioning of Cnp1 nucleosomes, a large portion of the endogenous centromere is dispensable for its essential activity in mediating chromosome segregation. We present biochemical evidence that Cnp1 occupancy directly correlates with silencing of the underlying reporter genes. Furthermore, using a newly developed pedigree analysis assay, we demonstrated the epigenetic inheritance of Cnp1 positioning and quantified the rate of occasional repositioning of Cnp1 nucleosomes throughout cell generations. Together, our results reveal the plasticity and the epigenetically inheritable nature of centromeric chromatin organization.

Dual mechanisms regulate the recruitment of spindle assembly checkpoint proteins to the budding yeast kinetochore

Quantitative knowledge of the recruitment of spindle assembly checkpoint (SAC) proteins by the kinetochore is essential to understanding the mechanisms that regulate protein recruitment and hence the strength of the SAC. Here this recruitment is quantified, and novel mechanisms are identified that strongly modulate SAC protein recruitment by the kinetochore.

Adaptor Autoregulation Promotes Coordinated Binding within Clathrin Coats

Background: There are multiple interacting clathrin adaptors at the trans-Golgi network and endosomes in yeast. Results: Autoregulation of one adaptor, Gga2, alters the temporal delay between recruitment of Gga2 and a second adaptor, Ent5, to organelles. Conclusion: The interaction between Gga2 and Ent5 is regulated by an autoregulatory sequence. Significance: This autoregulatory mechanism may ensure accurate membrane traffic in vivo. Membrane traffic is an essential process that allows protein and lipid exchange between the endocytic, lysosomal, and secretory compartments. Clathrin-mediated traffic between the trans-Golgi network and endosomes mediates responses to the environment through the sorting of biosynthetic and endocytic protein cargo. Traffic through this pathway is initiated by the controlled assembly of a clathrin-adaptor protein coat on the cytosolic surface of the originating organelle. In this process, clathrin is recruited by different adaptor proteins that act as a bridge between clathrin and the transmembrane cargo proteins to be transported. Interactions between adaptors and clathrin and between different types of adaptors lead to the formation of a densely packed protein network within the coat. A key unresolved issue is how the highly complex adaptor-clathrin interaction and adaptor-adaptor interaction landscape lead to the correct spatiotemporal assembly of the clathrin coat. Here we report the discovery of a new autoregulatory motif within the clathrin adaptor Gga2 that drives synergistic binding of Gga2 to clathrin and the adaptor Ent5. This autoregulation influences the temporal and/or spatial location of the Gga2-Ent5 interaction. We propose that this synergistic binding provides built-in regulation to ensure the correct assembly of clathrin coats.

Using protein dimers to maximize the protein hybridization efficiency with multisite DNA origami scaffolds

DNA origami provides a versatile platform for conducting ‘architecture-function’ analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.

A Sensitized Emission Based Calibration of FRET Efficiency for Probing the Architecture of Macromolecular Machines

Macromolecular machines participate in almost every cell biological function. These machines can take the form of well-defined protein structures such as the kinetochore, or more loosely organized protein assemblies like the endocytic coat. The protein architecture of these machines—the arrangement of multiple copies of protein subunits at the nanoscale, is necessary for understanding their cell biological function and biophysical mechanism. Defining this architecture in vivo presents a major challenge. High density of protein molecules within macromolecular machines severely limits the effectiveness of super-resolution microscopy. However, this density is ideal for Forster Resonance Energy Transfer (FRET), which can determine the proximity between neighboring molecules. Here, we present a simple FRET quantitation scheme that calibrates a standard epifluorescence microscope for measuring donor–acceptor separations. This calibration can be used to deduce FRET efficiency fluorescence intensity measurements. This method will allow accurate determination of FRET efficiency over a wide range of values and FRET pair number. It will also allow dynamic FRET measurements with high spatiotemporal resolution under cell biological conditions. Although the poor maturation efficiency of genetically encoded fluorescent proteins presents a challenge, we show that its effects can be alleviated. To demonstrate this methodology, we probe the in vivo architecture of the γ-Tubulin Ring. Our technique can be applied to study the architecture and dynamics of a wide range of macromolecular machines.

How the kinetochore switches off the spindle assembly checkpoint

The eukaryotic kinetochore senses whether or not it is attached to spindle microtubules. If unattached, it activates the signaling cascade of the Spindle Assembly Checkpoint (SAC) and delays cell division. The biochemistry of SAC signaling is well-understood. However, the molecular mechanism that couples SAC activation and inactivation to the attachment state of the kinetochore has been an enduring question in cell biology. Three recent studies address this question, but propose 2 different mechanisms, one mechanical in nature and the other based on biochemical competition. Here we highlight key results that led us to the ‘mechanical switch’ model, and propose that the SAC is silenced by a hybrid mechanism that uses biochemical competition as well as a mechanical switch. An unattached kinetochore initiates SAC signaling by enabling the phosphorylation of the kinetochore protein Spc105/KNL-1 by Mps1 kinase. This allows the kinetochore to recruit SAC proteins and generate the ‘wait-anaphase’ signal. After the kinetochore forms end-on microtubule attachment, it sheds SAC proteins and stops signaling. Two types of mechanisms can explain the attachment-induced removal of SAC proteins from the kinetochore. Microtubule attachment may interfere with the binding of one or more SAC proteins either by creating steric hindrance or by competing for a common binding site in the kinetochore. Alternatively, it may disrupt protein interactions mechanically, by changing protein organization in the kinetochore. To understand the mechanism of SAC silencing, we investigated the relatively simple budding yeast kinetochore that has a well-defined protein organization. We found that the yeast kinetochore encodes a microtubule-operated mechanical switch to control the SAC. In a mechanical switch, the closing of 2 terminals allows electricity to flow, while opening them stops this flow. In a similar manner, the Calponin-Homology domains of the Ndc80 complex and the phosphodomain of Spc105 operate as 2 protein terminals in the yeast kinetochore. They come together when the kinetochore is unattached, allowing Mps1 bound to the Calponin-Homology domains to phosphorylate Spc105 and activate the SAC (Fig. 1, top). Because these domains contain microtubule-binding sites, end-on microtubule attachment separates them by ~30 nm (Fig. 1, bottom). Now, Mps1 can no longer phosphorylate Spc105, and the SAC is inactivated. Thus, the nanoscale protein organization of the end-on kinetochore-microtubule attachment serves as the mechanical signal for SAC silencing. The signaling role for the kinetochore architecture is based on 4 key results. First, we found that microtubule attachment to the kinetochore must disrupt the interaction between Mps1 and Spc105 to silence the SAC; if this interaction is forced to occur, SAC protein recruitment occurs even if the kinetochore is attached. Second, attachment does not disrupt the Mps1-Spc105 interaction simply by removing Mps1 from the kinetochore. A residual pool of Mps1 persists at the kinetochore even after the formation of end-on microtubule attachment. Third, where Mps1 binds in the yeast kinetochore is critical to the ability of the kinetochore to inactivate the SAC after it forms end-on