Pericentrin is a Kinesin-1 Activator that Drives Centriole Motility

Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosome nucleated microtubules with motor proteins. However, it remains unknown how centrioles migrate in cellular contexts in which centrioles do not nucleate microtubules. Here, we demonstrate that during interphase inactive centrioles move directly along the noncentrosomal microtubule network as cargo for the motor protein Kinesin-1. We identify Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. PLP directly interacts with the cargo binding domain of Kinesin-1 and they comigrate on microtubules in vitro. Finally, we demonstrate that PLP-Kinesin-1 dependent transport is essential for centrosome asymmetry age dependent centrosome inheritance in asymmetric stem cell division.

New cell biological explanations for kinesin-linked axon degeneration

Axons are the slender, up to meter-long projections of neurons that form the biological cables wiring our bodies. Most of these delicate structures must survive for an organism's lifetime, meaning up to a century in humans. Axon maintenance requires life-sustaining motor protein-driven transport distributing materials and organelles from the distant cell body. It seems logic that impairing this transport causes systemic deprivation linking to axon degeneration. But the key steps underlying these pathological processes are little understood. To investigate mechanisms triggered by motor protein aberrations, we studied more than 40 loss- and gain-of-function conditions of motor proteins, cargo linkers or further genes involved in related processes of cellular physiology. We used one standardised Drosophila primary neuron system and focussed on the organisation of axonal microtubule bundles as an easy to assess readout reflecting axon integrity. We found that bundle disintegration into curled microtubules is caused by the losses of Dynein heavy chain and the Kif1 and Kif5 homologues Unc-104 and Kinesin heavy chain (Khc). Using point mutations of Khc and functional loss of its linker proteins, we studied which of Khc's sub-functions might link to microtubule curling. One cause was emergence of harmful reactive oxygen species through loss of Milton/Miro-mediated mitochondrial transport. In contrast, loss of the Kinesin light chain linker caused microtubule curling through an entirely different mechanism appearing to involve increased mechanical challenge to microtubule bundles through de-inhibition of Khc. The wider implications of our findings for the understanding of axon maintenance and pathology are discussed.

Microtubule Lattice Spacing Governs Cohesive Envelope Formation of Tau Family Proteins

Tau is an intrinsically-disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive envelopes around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here, we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau-envelope disassembly. Investigating other members of the tau-MAP family, we find MAP2 similarly forms envelopes governed by lattice-spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct segments. We conclude that the interdependent allostery between lattice-spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2.

MFN2 Deficiency Impairs Mitochondrial Transport and Downregulates Motor Protein Expression in Human Spinal Motor Neurons

Charcot-Marie-Tooth (CMT) disease is one of the most common genetically inherited neurological disorders and CMT type 2A (CMT 2A) is caused by dominant mutations in the mitofusin-2 (MFN2) gene. MFN2 is located in the outer mitochondrial membrane and is a mediator of mitochondrial fusion, with an essential role in maintaining normal neuronal functions. Although loss of MFN2 induces axonal neuropathy, the detailed mechanism by which MFN2 deficiency results in axonal degeneration of human spinal motor neurons remains largely unknown. In this study, we generated MFN2-knockdown human embryonic stem cell (hESC) lines using lentivirus expressing MFN2 short hairpin RNA (shRNA). Using these hESC lines, we found that MFN2 loss did not affect spinal motor neuron differentiation from hESCs but resulted in mitochondrial fragmentation and dysfunction as determined by live-cell imaging. Notably, MFN2-knockodwn spinal motor neurons exhibited CMT2A disease-related phenotypes, including extensive perikaryal inclusions of phosphorylated neurofilament heavy chain (pNfH), frequent axonal swellings, and increased pNfH levels in long-term cultures. Importantly, MFN2 deficit impaired anterograde and retrograde mitochondrial transport within axons, and reduced the mRNA and protein levels of kinesin and dynein, indicating the interfered motor protein expression induced by MFN2 deficiency. Our results reveal that MFN2 knockdown induced axonal degeneration of spinal motor neurons and defects in mitochondrial morphology and function. The impaired mitochondrial transport in MFN2-knockdown spinal motor neurons is mediated, at least partially, by the altered motor proteins, providing potential therapeutic targets for rescuing axonal degeneration of spinal motor neurons in CMT2A disease.

Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD

Efficient control of transcription is essential in bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. Long-lasting stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD represents a new class of ATP-dependent motor protein distantly related to helicases. It was first identified in the Gram-positive model Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram-positive bacteria. In this work we have undertaken the first comprehensive investigation of the phylogenetic diversity of HelD proteins. We show that genes in certain classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram-negative bacteria. HelD proteins represent an important new class of transcription factor associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility

The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.

Analysis of the Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein-1 Using a Chemical “Toolkit”

Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure–activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues’ effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the “glutamate switch” mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.