Cytoskeletal motor proteins are biological nanomachines that convert chemical energy into mechanical work to carry out various functions such as cell division, cell motility, cargo transport, muscle contraction, beating of cilia and flagella, and ciliogenesis. Most of these processes are driven by the collective operation of several motors in the crowded viscous intracellular environment. Imaging and manipulation of the motors with powerful experimental probes have been complemented by mathematical analysis and computer simulations of the corresponding theoretical models. In this article, we illustrate some of the key theoretical approaches used to understand how coordination, cooperation and competition of multiple motors in the crowded intra-cellular environment drive the processes that are essential for biological function of a cell. In spite of the focus on theory, experimentalists will also find this article as an useful summary of the progress made so far in understanding multiple motor systems.

Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretion which contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis.

OBJECTIVE: DYNC1H1 variants are involved on a disease spectrum from neuromuscular disorders to neurodevelopmental disorders. DYNC1H1-related epilepsy has been reported in small cohorts. We dissect the electroclinical features of 34 patients harboring de novo DYNC1H1 pathogenic variants, identify subphenotypes on the DYNC1H1-related epilepsy spectrum, and compare the genotype-phenotype correlations observed in our cohort with the literature.
METHODS: Patients harboring de novo DYNC1H1 pathogenic variants were recruited through international collaborations. Clinical data were retrospectively collected. Latent class analysis was performed to identify subphenotypes. Multivariable binary logistic regression analysis was applied to investigate the association with DYNC1H1 protein domains.
RESULTS: DYNC1H1-related epilepsy presented with infantile epileptic spasms syndrome (IESS) in 17 subjects (50%), and in 25% of these individuals the epileptic phenotype evolved into Lennox-Gastaut syndrome (LGS). In 12 patients (35%), focal onset epilepsy was defined. In two patients, the epileptic phenotype consisted of generalized myoclonic epilepsy, with a progressive phenotype in one individual harboring a frameshift variant. In approximately 60% of our cohort, seizures were drug-resistant. Malformations of cortical development were noticed in 79% of our patients, mostly on the lissencephaly-pachygyria spectrum, particularly with posterior predominance in a half of them. Midline and infratentorial abnormalities were additionally reported in 45% and 27% of subjects. We have identified three main classes of subphenotypes on the DYNC1H1-related epilepsy spectrum.
CONCLUSIONS: We propose a classification in which pathogenic de novo DYNC1H1 variants feature drug-resistant IESS in half of cases with potential evolution to LGS (Class 1), developmental and epileptic encephalopathy other than IESS and LGS (Class 2), or less severe focal or genetic generalized epilepsy including a progressive phenotype (Class 3). We observed an association between stalk domain variants and Class 1 phenotypes. The variants p.Arg309His and p.Arg1962His were common and associated with Class 1 subphenotype in our cohort. These findings may aid genetic counseling of patients with DYNC1H1-related epilepsy.

BACKGROUND: Dysfunction of motile cilia, including respiratory cilia and sperm flagella, typically leads to primary ciliary dyskinesia and male infertility or low fertility in humans. Genetic defects of LRRC6 have been associated with primary ciliary dyskinesia and asthenozoospermia due to abnormal ultrastructure of ciliated axonemes.
OBJECTIVE: To identify novel mutations of the LRRC6 gene related to multiple morphological abnormalities of the sperm flagella and male infertility and investigate the underlying molecular mechanisms involved.
METHODS: The LRRC6 mutations were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning, and transmission electron microscopy were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Further tandem mass tagging proteomics analyses were performed to explore the effect of mutations and confirmed by immunostaining and western blotting. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic LRRC6 mutations.
RESULTS: In this study, we identified a novel homozygous LRRC6 mutation in a consanguineous family, characterized by asthenozoospermia and primary ciliary dyskinesia. Further Semen parameter and morphology analysis demonstrate that the novel LRRC6 mutation leads to a significant reduction in sperm flagella length, a decrease in sperm progressive motility parameters, and abnormalities of sperm ultrastructure. Specifically, the absence of outer dynein arms and inner dynein arms, and incomplete mitochondrial sheath in the flagellar mid-piece were observed by transmission electron microscopy. In addition, tandem mass tagging proteomics analysis revealed that spermatozoa obtained from patients harboring the LRRC6 mutation exhibited a significant decrease in the expression levels of proteins related to the assembly and function of dynein axonemal arms. Functional analysis revealed that this novel LRRC6 mutation disrupted the function of the leucine-rich repeat containing 6 protein, which in turn affects the expression of the dynein arm proteins and leucine-rich repeat containing 6-interacting proteins CCDC40, SPAG1, and ZMYND10. Finally, we reported a successful pregnancy through assisted reproductive technology with intracytoplasmic sperm injection in the female partner of the proband.
CONCLUSIONS: This study highlights the identification of a novel homozygous LRRC6 mutation in a consanguineous family and its impact on sperm progressive motility, morphology, and sperm kinetics parameters, which could facilitate the genetic diagnosis of asthenozoospermia and offer valuable perspectives for future genetic counseling endeavors.

Mechanical stress significantly affects the physiological functions of cells, including tissue homeostasis, cytoskeletal alterations, and intracellular transport. As a major cytoskeletal component, microtubules respond to mechanical stimulation by altering their alignment and polymerization dynamics. Previously, we reported that microtubules may modulate cargo transport by one of the microtubule-associated motor proteins, dynein, under compressive mechanical stress. Despite the critical role of tensile stress in many biological functions, how tensile stress on microtubules regulates cargo transport is yet to be unveiled. The present study demonstrates that the low-level tensile stress-induced microtubule deformation facilitates dynein-driven transport. We validate our experimental findings using all-atom molecular dynamics simulation. Our study may provide important implications for developing new therapies for diseases that involve impaired intracellular transport.

Cytoplasmic dynein-1 (dynein) is a microtubule-associated, minus end-directed motor that traffics hundreds of different cargos. Dynein must discriminate between cargos and traffic them at the appropriate time from the correct cellular region. How dynein\'s trafficking activity is regulated in time or cellular space remains poorly understood. Here, we identify CCSer2 as the first known protein to gate dynein activity in the spatial dimension. CCSer2 promotes the migration of developing zebrafish primordium cells and of cultured human cells by facilitating the trafficking of cargos that are acted on by cortically localized dynein. CCSer2 inhibits the interaction between dynein and its regulator Ndel1 exclusively at the cell periphery, resulting in localized dynein activation. Our findings suggest that the spatial specificity of dynein is achieved by the localization of proteins that disinhibit Ndel1. We propose that CCSer2 defines a broader class of proteins that activate dynein in distinct microenvironments via Ndel1 inhibition.

Lysosomes are dynamic cellular structures that adaptively remodel their membrane in response to stimuli, including membrane damage. We previously uncovered a process we term LYTL (LYsosomal Tubulation/sorting driven by Leucine-Rich Repeat Kinase 2 [LRRK2]), wherein damaged lysosomes generate tubules sorted into mobile vesicles. LYTL is orchestrated by the Parkinson\'s disease-associated kinase LRRK2 that recruits the motor adaptor protein and RHD family member JIP4 to lysosomes via phosphorylated RAB proteins. To identify new players involved in LYTL, we performed unbiased proteomics on isolated lysosomes after LRRK2 kinase inhibition. Our results demonstrate that there is recruitment of RILPL1 to ruptured lysosomes via LRRK2 activity to promote phosphorylation of RAB proteins at the lysosomal surface. RILPL1, which is also a member of the RHD family, enhances the clustering of LRRK2-positive lysosomes in the perinuclear area and causes retraction of LYTL tubules, in contrast to JIP4 which promotes LYTL tubule extension. Mechanistically, RILPL1 binds to p150Glued, a dynactin subunit, facilitating the transport of lysosomes and tubules to the minus end of microtubules. Further characterization of the tubulation process revealed that LYTL tubules move along tyrosinated microtubules, with tubulin tyrosination proving essential for tubule elongation. In summary, our findings emphasize the dynamic regulation of LYTL tubules by two distinct RHD proteins and pRAB effectors, serving as opposing motor adaptor proteins: JIP4, promoting tubulation via kinesin, and RILPL1, facilitating tubule retraction through dynein/dynactin. We infer that the two opposing processes generate a metastable lysosomal membrane deformation that facilitates dynamic tubulation events.

Herpesvirus assembly requires the cytoplasmic association of large macromolecular and membrane structures that derive from both the nucleus and cytoplasmic membrane systems. Results from the study of human cytomegalovirus (HCMV) in cells where it organizes a perinuclear cytoplasmic virus assembly compartment (cVAC) show a clear requirement for the minus-end-directed microtubule motor, dynein, for virus assembly. In contrast, the assembly of herpes simplex virus -1 (HSV-1) in epithelial cells where it forms multiple dispersed, peripheral assembly sites is only mildly inhibited by the microtubule-depolymerizing agent, nocodazole. Here, we make use of a neuronal cell line system in which HSV-1 forms a single cVAC and show that dynein and its co-factor dynactin localize to the cVAC, and dynactin is associated with membranes that contain the virion tegument protein pUL11. We also show that the virus membrane-associated structural proteins pUL51 and the viral envelope glycoprotein gE arrive at the cVAC by different routes. Specifically, gE arrives at the cVAC after retrieval from the plasma membrane, suggesting the need for an intact retrograde transport system. Finally, we demonstrate that inhibition of dynactin function profoundly inhibits cVAC formation and virus production during the cytoplasmic assembly phase of infection.IMPORTANCEMany viruses reorganize cytoplasmic membrane systems and macromolecular transport systems to promote the production of progeny virions. Clarifying the mechanisms by which they accomplish this may reveal novel therapeutic strategies and illustrate mechanisms that are critical for normal cellular organization. Here, we explore the mechanism by which HSV-1 moves macromolecular and membrane cargo to generate a virus assembly compartment in the infected cell. We find that the virus makes use of a well-characterized, microtubule-based transport system that is stabilized against drugs that disrupt microtubules.

The forces that orient the spindle in human cells remain poorly understood due to a lack of direct mechanical measurements in mammalian systems. We use magnetic tweezers to measure the force on human mitotic spindles. Combining the spindle\'s measured resistance to rotation, the speed at which it rotates after laser ablating astral microtubules, and estimates of the number of ablated microtubules reveals that each microtubule contacting the cell cortex is subject to ∼5 pN of pulling force, suggesting that each is pulled on by an individual dynein motor. We find that the concentration of dynein at the cell cortex and extent of dynein clustering are key determinants of the spindle\'s resistance to rotation, with little contribution from cytoplasmic viscosity, which we explain using a biophysically based mathematical model. This work reveals how pulling forces on astral microtubules determine the mechanics of spindle orientation and demonstrates the central role of cortical dynein clustering.

The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that have a specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immunolabeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the three-dimensional ultrastructure of the cilium. Here, we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT)88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immunolabeling and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.