Kinesin-1 is a dynamic heterotetrameric assembly of two heavy and two light chains (KHC and KLC) that mediates microtubule-based intracellular transport of many different cargoes. The complex adopts a compact, autoinhibited state that is activated by cargo-adaptor proteins containing specific short linear peptide motifs (SLiMs). These motifs interact with the tetratricopeptide repeat (TPR) domains of the KLCs. The mechanism coupling SLiM recognition to activation-associated conformational changes in the complex is unknown. Here we combine protein design, computational modelling, biophysical analysis, and electron microscopy to examine the structural and mechanistic consequences of SLiM binding to the KLC-TPR domain within the complete heterotetrameric holoenzyme. We show that coiled coil 1 (CC1) of the KHC docks KLC TPR domains in the autoinhibited complex, forming the ‘shoulder’ feature observed in electron microscopy. Disrupting this interaction or binding an activating SLiM dislocates the TPR shoulder, freeing the motor domains and promoting transition between its closed, inactive, and open states. Opening the kinesin-1 complex facilitates binding to the microtubule-associated kinesin-1 cofactor, MAP7. Therefore, cargo-mediated dislocation of the TPR shoulder serves as a key initial step in kinesin-1 activation, allosterically linking cargo binding to motor dynamics.

Cells depend on a complex and precisely regulated subcellular organization, largely driven by the cytoskeleton and motor proteins that control intracellular transport. This review explores innovative strategies to manipulate cellular architecture using targeted protein design and engineering of cytoskeletal elements and molecular motors. We highlight advances in inducible dimerization techniques, which enable precise control over cytoskeletal dynamics through light- and small-molecule-sensitive domains. In addition, we discuss modifications to motor proteins that alter directionality, processivity, and cargo specificity, providing insights into their roles in cellular transport. Rapid advances in de novo protein design offer new tools to hijack natural cytoskeletal machinery and create synthetic elements for cellular architecture, including membraneless organelles and synthetic cytoskeletal tracks. This research promises to deepen our understanding of cellular organization, uncover regulatory mechanisms, and provide new proteins for therapeutic applications and synthetic cell development.

Kinesin-1 is a dynamic heterotetrameric assembly of two heavy and two light chains (KHC and KLC) that mediates microtubule-based intracellular transport of many different cargoes. The complex adopts a compact, autoinhibited state that is activated by cargo-adaptor proteins containing specific short linear peptide motifs (SLiMs). These motifs interact with the tetratricopeptide repeat (TPR) domains of the KLCs. The mechanism coupling SLiM recognition to activation-associated conformational changes in the complex is unknown. Here we combine protein design, computational modelling, biophysical analysis, and electron microscopy to examine the structural and mechanistic consequences of SLiM binding to the KLC-TPR domain within the complete heterotetrameric holoenzyme. We show that coiled coil 1 (CC1) of the KHC docks KLC TPR domains in the autoinhibited complex, forming the “shoulder” feature observed in electron microscopy. Disrupting this interaction or binding an activating SLiM dislocates the TPR shoulder, freeing the motor domains and promoting transition between its closed, inactive, and open states. Therefore, cargo-mediated dislocation of the TPR shoulder serves as a key initial step in kinesin-1 activation, allosterically linking cargo binding to motor dynamics. ### Competing Interest Statement The authors have declared no competing interest.

Mitochondria are the powerhouses of eukaryotic cells, composed mostly of nuclear-encoded proteins imported from the cytosol. Thus, problems with the import machinery will disrupt their regenerative capacity and the cell’s energy supplies – particularly troublesome for energy-demanding cells of nervous tissue and muscle. Unsurprisingly then, import breakdown is implicated in disease. Here, we explore the consequences of import failure in mammalian cells; wherein, blocking the import machinery impacts mitochondrial ultra-structure and dynamics, but, surprisingly, does not affect import. Our data are consistent with a response involving intercellular mitochondrial transport via tunnelling nanotubes to import healthy mitochondria and jettison those with blocked import sites. These observations support the existence of a widespread mechanism for the rescue of mitochondrial dysfunction.

Many enzymes are allosterically regulated via conformational change; however, our ability to manipulate these structural changes and control function is limited. Here we install a conformational switch for allosteric activation into the kinesin-1 microtubule motor in vitro and in cells. Kinesin-1 is a heterotetramer that accesses open active and closed autoinhibited states. The equilibrium between these states centers on a flexible elbow within a complex coiled-coil architecture. We target the elbow to engineer a closed state that can be opened with a de novo designed peptide. The alternative states are modeled computationally and confirmed by biophysical measurements and electron microscopy. In cells, peptide-driven activation increases kinesin transport, demonstrating a primary role for conformational switching in regulating motor activity. The designs are enabled by our understanding of ubiquitous coiled-coil structures, opening possibilities for controlling other protein activities.

Crosstalk between the actin and microtubule cytoskeletons is important for many cellular processes. Recent studies have shown that microtubules and F-actin can assemble to form a composite structure where F-actin occupies the microtubule lumen. Whether these cytoskeletal hybrids exist in physiological settings and how they are formed is unclear. Here, we show that the short-crossover Class I actin filament previously identified inside microtubules in human HAP1 cells is cofilin-bound F-actin. Lumenal F-actin can be reconstituted in vitro, but cofilin is not essential. Moreover, actin filaments with both cofilin-bound and canonical morphologies reside within human platelet microtubules under physiological conditions. We propose that stress placed upon the microtubule network during motor-driven microtubule looping and sliding may facilitate the incorporation of actin into microtubules.

AbstractMany enzymes are allosterically regulated. Our ability to manipulate these structural changes is limited. Here we install an allosteric switch into the kinesin-1 microtubule motorin vitroand in cells. Kinesin-1 is a heterotetramer that accesses open active and closed auto-inhibited states. The equilibrium between these centres on a flexible elbow within a complex coiled-coil architecture. We target the elbow to engineer a closed state that can be opened with ade novodesigned peptide. The alternative states are modelled computationally and confirmed by biophysical measurements and electron microscopy. In cells, peptide-driven activation increases kinesin transport, demonstrating a primary role for conformational switching in regulating motor activity. The designs are enabled by our understanding of ubiquitous coiled-coil structures, opening possibilities for controlling other protein activities.One Sentence SummaryDe novopeptide and protein design are used to engineer an allosteric switch into kinesin-1 motorsin vitroand directly in cells.

Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the α-helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative-stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1 in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drives kinesin-1 activation.

Mitochondria are the powerhouses of eukaryotic cells, composed mostly of nuclear-encoded proteins imported from the cytosol. Thus, problems with the import machinery will disrupt their regenerative capacity and the cell’s energy (ATP) supplies–particularly troublesome for energy demanding cells like neurons and myocytes. Unsurprisingly then, dysfunctional import is implicated in disease. This study explores the consequences of import failure in mammalian cells; wherein, blocking the import machinery has profound effects on mitochondrial ultra-structure and dynamics, but, surprisingly, does not impact import. The explanation is an astonishing response involving intercellular mitochondrial transfer via tunnelling nanotubes: for the import of healthy mitochondria and jettisoning of those with jammed import sites. These observations support the existence of a widespread mechanism for the rescue of mitochondrial protein import failure. One-Sentence Summary A mitochondrial import rescue mechanism involving intercellular mitochondrial transport through tunneling nanotubes (TNTs).

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

The lumen of cytoplasmic microtubules is a poorly explored expanse of intracellular space. Although typically represented in textbooks as a hollow tube, studies over several decades have shown that the microtubule lumen is occupied by a range of morphologically diverse components. These are predominantly globular particles of varying sizes which appear to exist either in isolation, bind to the microtubule wall, or form discontinuous columns that extend through the lumenal space. Actin filaments with morphologies distinct from the canonical cytoplasmic forms have also now been found within the microtubule lumen. In this review, we examine the historic literature that observed these lumenal components in tissues from diverse species and integrate it with recent cryo-electron tomography studies that have begun to identify lumenal proteins. We consider their cell and tissue distribution, possible mechanisms of incorporation, and potential functions. It is likely that continuing work in this area will open a new frontier in cytoskeletal biology.

Increasingly, it is possible to design peptide and protein assemblies de novo from first principles or computationally. This approach provides new routes to functional synthetic polypeptides, including designs to target and bind proteins of interest. Much of this work has been developed in vitro. Therefore, a challenge is to deliver de novo polypeptides efficiently to sites of action within cells. Here we describe the design, characterisation, intracellular delivery, and subcellular localisation of a de novo synthetic peptide system. This system comprises a dual-function basic peptide, programmed both for cell penetration and target binding, and a complementary acidic peptide that can be fused to proteins of interest and introduced into cells using synthetic DNA. The designs are characterised in vitro using biophysical methods and X-ray crystallography. The utility of the system for delivery into mammalian cells and subcellular targeting is demonstrated by marking organelles and actively engaging functional protein complexes.

The prototypic and ubiquitous microtubule motor, kinesin-1, uses a variety of adaptor proteins to facilitate the selective transport of diverse cargo within the cell. These cargo adaptors bind to the motor complex through interactions with the kinesin light or heavy chains (KLCs or KHCs). In this issue of Genes & Development, Dimitrova-Paternoga et al. (pp. 976-991) present the first structural characterization of a KHC-cargo adaptor interface. They describe an antiparallel heterotrimeric coiled-coil complex between the carboxy tail of KHC and Tm1-I/C (aTm1), the atypical tropomyosin that is important for oskar mRNA transport in Drosophila oocytes. This interaction enhances direct binding between KHC and RNA. Their findings demonstrate the structural plasticity of the KHC tail as a platform for protein-protein interactions and reveal how a cargo adaptor protein can modify a motor-RNA interface to promote transport.

The cargo-binding capabilities of cytoskeletal motor proteins have expanded during evolution through both gene duplication and alternative splicing. For the light chains of the kinesin-1 family of microtubule motors, this has resulted in an array of carboxyl-terminal domain sequences of unknown molecular function. Here, combining phylogenetic analyses with biophysical, biochemical, and cell biology approaches, we identify a highly conserved membrane-induced curvature-sensitive amphipathic helix within this region of a subset of long kinesin light-chain paralogs and splice isoforms. This helix mediates the direct binding of kinesin-1 to lipid membranes. Membrane binding requires specific anionic phospholipids, and it contributes to kinesin-1-dependent lysosome positioning, a canonical activity that, until now, has been attributed exclusively the recognition of organelle-associated cargo adaptor proteins. This leads us to propose a protein-lipid coincidence detection framework for kinesin-1-mediated organelle transport.

Synthetic peptides are attractive candidates to manipulate protein-protein interactions inside the cell as they mimic natural interactions to compete for binding. However, protein-peptide interactions are often dynamic and weak. A challenge is to design peptides that make improved interactions with the target. Here, we devise a fragment-linking strategy-"mash-up" design-to deliver a high-affinity ligand, KinTag, for the kinesin-1 motor. Using structural insights from natural micromolar-affinity cargo-adaptor ligands, we have identified and combined key binding features in a single, high-affinity ligand. An X-ray crystal structure demonstrates interactions as designed and reveals only a modest increase in interface area. Moreover, when genetically encoded, KinTag promotes transport of lysosomes with higher efficiency than natural sequences, revealing a direct link between motor-adaptor binding affinity and organelle transport. Together, these data demonstrate a fragment-linking strategy for peptide design and its application in a synthetic motor ligand to direct cellular cargo transport.

Microtubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organisation to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryo-electron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small-molecule induced, microtubule-based cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in-situ study of microtubule structure and contents.

Centrosomes are composed of two orthogonally arranged centrioles surrounded by an electron-dense matrix called the pericentriolar material (PCM). Centrioles are cylinders with diameters of ~250 nm, are several hundred nanometres in length and consist of 9-fold symmetrically arranged microtubules (MT). In dividing animal cells, centrosomes act as the principal MT-organising centres and they also organise actin, which tunes cytoplasmic MT nucleation. In some specialised cells, the centrosome acquires additional critical structures and converts into the base of a cilium with diverse functions including signalling and motility. These structures are found in most eukaryotes and are essential for development and homoeostasis at both cellular and organism levels. The ultrastructure of centrosomes and their derived organelles have been known for more than half a century. However, recent advances in a number of techniques have revealed the high-resolution structures (at Å-to-nm scale resolution) of centrioles and have begun to uncover the molecular principles underlying their properties, including: protein components; structural elements; and biogenesis in various model organisms. This review covers advances in our understanding of the features and processes that are critical for the biogenesis of the evolutionarily conserved structures of the centrosomes. Furthermore, it discusses how variations of these aspects can generate diversity in centrosome structure and function among different species and even between cell types within a multicellular organism.

Cytoskeletal motors of the dynein, kinesin and myosin superfamilies maintain and adapt subcellular organelle organization to meet functional demands and support the vesicular transport of material between organelles. These motors require the capacity to specifically recognize the vesicle/organelle to be transported and are capable of selective recognition of multiple cargo. Recent studies have begun to uncover the molecular basis for motor recruitment and have highlighted the role of organelle-associated 'cargo-adaptor' proteins in cellular transport. These adaptors possess sequences and/or structural features that enable both motor recruitment and activation from regulated, inactive, states to enable motility on the cytoskeleton. Motor-cargo adaptor interactions define a key organelle-cytoskeleton interface, acting as crucial regulatory hubs to enable the cell to finely control membrane trafficking and organelle dynamics. Understanding the molecular basis of these interactions may offer new opportunities to control and manipulate cytoskeletal and organelle dynamics for the development of new research tools and potentially therapeutics.