Conduction-Dominated Cryomesh for Organism Vitrification.

Vitrification-based cryopreservation is a promising approach to achieving long-term storage of biological systems for maintaining biodiversity, healthcare, and sustainable food production. Using the "cryomesh" system achieves rapid cooling and rewarming of biomaterials, but further improvement in cooling rates is needed to increase biosystem viability and the ability to cryopreserve new biosystems. Improved cooling rates and viability are possible by enabling conductive cooling through cryomesh. Conduction-dominated cryomesh improves cooling rates from twofold to tenfold (i.e., 0.24 to 1.2 × 105  °C min-1 ) in a variety of biosystems. Higher thermal conductivity, smaller mesh wire diameter and pore size, and minimizing the nitrogen vapor barrier (e.g., vertical plunging in liquid nitrogen) are key parameters to achieving improved vitrification. Conduction-dominated cryomesh successfully vitrifies coral larvae, Drosophila embryos, and zebrafish embryos with improved outcomes. Not only a theoretical foundation for improved vitrification in µm to mm biosystems but also the capability to scale up for biorepositories and/or agricultural, aquaculture, or scientific use are demonstrated.

Early-phase drug discovery of ß-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia type 5.

ß-III-Spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 L253P mutation in the cytoskeletal protein ß-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small-molecule actin-binding modulators of the L253P mutant ß-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P ß-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for ß-III-spectrin ABD modulators.

ß-III-spectrin N-terminus is required for high-affinity actin binding and SCA5 neurotoxicity.

Recent structural studies of ß-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem calponin homology domains (CH1 and CH2). Here we investigated in Drosophila the significance of the ß-spectrin N-terminus, and explored its functional interaction with a CH2-localized L253P mutation that underlies the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). We report that pan-neuronal expression of an N-terminally truncated ß-spectrin fails to rescue lethality resulting from a ß-spectrin loss-of-function allele, indicating that the N-terminus is essential to ß-spectrin function in vivo. Significantly, N-terminal truncation rescues neurotoxicity and defects in dendritic arborization caused by L253P. In vitro studies show that N-terminal truncation eliminates L253P-induced high-affinity actin binding, providing a mechanistic basis for rescue. These data suggest that N-terminal sequences may be useful therapeutic targets for small molecule modulation of the aberrant actin binding associated with SCA5 ß-spectrin and spectrin-related disease proteins.

Novel drug discovery platform for spinocerebellar ataxia, using fluorescence technology targeting ß-III-spectrin.

Numerous diseases are linked to mutations in the actin-binding domains (ABDs) of conserved cytoskeletal proteins, including ß-III-spectrin, α-actinin, filamin, and dystrophin. A ß-III-spectrin ABD mutation (L253P) linked to spinocerebellar ataxia type 5 (SCA5) causes a dramatic increase in actin binding. Reducing actin binding of L253P is thus a potential therapeutic approach for SCA5 pathogenesis. Here, we validate a high-throughput screening (HTS) assay to discover potential disrupters of the interaction between the mutant ß-III-spectrin ABD and actin in live cells. This assay monitors FRET between fluorescent proteins fused to the mutant ABD and the actin-binding peptide Lifeact, in HEK293-6E cells. Using a specific and high-affinity actin-binding tool compound, swinholide A, we demonstrate HTS compatibility with an excellent Z'-factor of 0.67 ± 0.03. Screening a library of 1280 pharmacologically active compounds in 1536-well plates to determine assay robustness, we demonstrate high reproducibility across plates and across days. We identified nine Hits that reduced FRET between Lifeact and ABD. Four of those Hits were found to reduce Lifeact cosedimentation with actin, thus establishing the potential of our assay for detection of actin-binding modulators. Concurrent to our primary FRET assay, we also developed a high-throughput compatible counter screen to remove undesirable FRET Hits. Using the FRET Hits, we show that our counter screen is sensitive to undesirable compounds that cause cell toxicity or ABD aggregation. Overall, our FRET-based HTS platform sets the stage to screen large compound libraries for modulators of ß-III-spectrin, or disease-linked spectrin-related proteins, for therapeutic development.

Kinesin-5 mediated chromosome congression in insect spindles.

INTRODUCTION: The microtubule motor protein kinesin-5 is well known to establish the bipolar spindle by outward sliding of antiparallel interpolar microtubules. In yeast, kinesin-5 also facilitates chromosome alignment "congression" at the spindle equator by preferentially depolymerizing long kinetochore microtubules (kMTs). The motor protein kinesin-8 has also been linked to chromosome congression. Therefore, we sought to determine whether kinesin-5 or kinesin-8 facilitates chromosome congression in insect spindles. METHODS: RNAi of the kinesin-5 Klp61F and kinesin-8 Klp67A were performed separately in Drosophila melanogaster S2 cells to test for inhibited chromosome congression. Klp61F RNAi, Klp67A RNAi, and control metaphase mitotic spindles expressing fluorescent tubulin and fluorescent Cid were imaged, and their fluorescence distributions were compared. RESULTS: RNAi of Klp61F with a weak Klp61F knockdown resulted in longer kMTs and less congressed kinetochores compared to control over a range of conditions, consistent with kinesin-5 length-dependent depolymerase activity. RNAi of the kinesin-8 Klp67A revealed that kMTs relative to the spindle lengths were not longer compared to control, but rather that the spindles were longer, indicating that Klp67A acts preferentially as a length-dependent depolymerase on interpolar microtubules without significantly affecting kMT length and chromosome congression. CONCLUSIONS: This study demonstrates that in addition to establishing the bipolar spindle, kinesin-5 regulates kMT length to facilitate chromosome congression in insect spindles. It expands on previous yeast studies, and it expands the role of kinesin-5 to include kMT assembly regulation in eukaryotic mitosis.

Structural basis for high-affinity actin binding revealed by a ß-III-spectrin SCA5 missense mutation.

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein ß-III-spectrin. Previously, a SCA5 mutation resulting in a leucine-to-proline substitution (L253P) in the actin-binding domain (ABD) was shown to cause a 1000-fold increase in actin-binding affinity. However, the structural basis for this increase is unknown. Here, we report a 6.9 Å cryo-EM structure of F-actin complexed with the L253P ABD. This structure, along with co-sedimentation and pulsed-EPR measurements, demonstrates that high-affinity binding caused by the CH2-localized mutation is due to opening of the two CH domains. This enables CH1 to bind actin aided by an unstructured N-terminal region that becomes α-helical upon binding. This helix is required for association with actin as truncation eliminates binding. Collectively, these results shed light on the mechanism by which ß-III-spectrin, and likely similar actin-binding proteins, interact with actin, and how this mechanism can be perturbed to cause disease.

ß-III-spectrin spinocerebellar ataxia type 5 mutation reveals a dominant cytoskeletal mechanism that underlies dendritic arborization.

A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the actin-binding domain (ABD) of ß-III-spectrin causes high-affinity actin binding and decreased thermal stability in vitro. Here we show in mammalian cells, at physiological temperature, that the mutant ABD retains high-affinity actin binding. Significantly, we provide evidence that the mutation alters the mobility and recruitment of ß-III-spectrin in mammalian cells, pointing to a potential disease mechanism. To explore this mechanism, we developed a Drosophila SCA5 model in which an equivalent mutant Drosophila ß-spectrin is expressed in neurons that extend complex dendritic arbors, such as Purkinje cells, targeted in SCA5 pathogenesis. The mutation causes a proximal shift in arborization coincident with decreased ß-spectrin localization in distal dendrites. We show that SCA5 ß-spectrin dominantly mislocalizes α-spectrin and ankyrin-2, components of the endogenous spectrin cytoskeleton. Our data suggest that high-affinity actin binding by SCA5 ß-spectrin interferes with spectrin-actin cytoskeleton dynamics, leading to a loss of a cytoskeletal mechanism in distal dendrites required for dendrite stabilization and arbor outgrowth.

A STRIPAK complex mediates axonal transport of autophagosomes and dense core vesicles through PP2A regulation.

Autophagy plays an essential role in the cellular homeostasis of neurons, facilitating the clearance of cellular debris. This clearance process is orchestrated through the assembly, transport, and fusion of autophagosomes with lysosomes for degradation. The motor protein dynein drives autophagosome motility from distal sites of assembly to sites of lysosomal fusion. In this study, we identify the scaffold protein CKA (connector of kinase to AP-1) as essential for autophagosome transport in neurons. Together with other core components of the striatin-interacting phosphatase and kinase (STRIPAK) complex, we show that CKA associates with dynein and directly binds Atg8a, an autophagosomal protein. CKA is a regulatory subunit of PP2A, a component of the STRIPAK complex. We propose that the STRIPAK complex modulates dynein activity. Consistent with this hypothesis, we provide evidence that CKA facilitates axonal transport of dense core vesicles and autophagosomes in a PP2A-dependent fashion. In addition, CKA-deficient flies exhibit PP2A-dependent motor coordination defects. CKA function within the STRIPAK complex is crucial to prevent transport defects that may contribute to neurodegeneration.

A human ß-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding.

Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein ß-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of ß-III-spectrin. We report that the L253P substitution in the isolated ß-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 µM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P ß-III-spectrin is a likely driver of neurodegeneration.

Coordination of autophagosome-lysosome fusion and transport by a Klp98A-Rab14 complex in Drosophila.

Degradation of cellular material by autophagy is essential for cell survival and homeostasis, and requires intracellular transport of autophagosomes to encounter acidic lysosomes through unknown mechanisms. Here, we identify the PX-domain-containing kinesin Klp98A as a new regulator of autophagosome formation, transport and maturation in Drosophila. Depletion of Klp98A caused abnormal clustering of autophagosomes and lysosomes at the cell center and reduced the formation of starvation-induced autophagic vesicles. Reciprocally, overexpression of Klp98A redistributed autophagic vesicles towards the cell periphery. These effects were accompanied by reduced autophagosome-lysosome fusion and autophagic degradation. In contrast, depletion of the conventional kinesin heavy chain caused a similar mislocalization of autophagosomes without perturbing their fusion with lysosomes, indicating that vesicle fusion and localization are separable and independent events. Klp98A-mediated fusion required the endolysosomal GTPase Rab14, which interacted and colocalized with Klp98A, and required Klp98A for normal localization. Thus, Klp98A coordinates the movement and fusion of autophagic vesicles by regulating their positioning and interaction with the endolysosomal compartment.

Methods to identify and analyze gene products involved in neuronal intracellular transport using Drosophila.

Proper neuronal function critically depends on efficient intracellular transport and disruption of transport leads to neurodegeneration. Molecular pathways that support or regulate neuronal transport are not fully understood. A greater understanding of these pathways will help reveal the pathological mechanisms underlying disease. Drosophila melanogaster is the premier model system for performing large-scale genetic functional screens. Here we describe methods to carry out primary and secondary genetic screens in Drosophila aimed at identifying novel gene products and pathways that impact neuronal intracellular transport. These screens are performed using whole animal or live cell imaging of intact neural tissue to ensure integrity of neurons and their cellular environment. The primary screen is used to identify gross defects in neuronal function indicative of a disruption in microtubule-based transport. The secondary screens, conducted in both motoneurons and dendritic arborization neurons, will confirm the function of candidate gene products in intracellular transport. Together, the methodologies described here will support labs interested in identifying and characterizing gene products that alter intracellular transport in Drosophila.

Relay of retrograde synaptogenic signals through axonal transport of BMP receptors.

Neuronal function depends on the retrograde relay of growth and survival signals from the synaptic terminal, where the neuron interacts with its targets, to the nucleus, where gene transcription is regulated. Activation of the Bone Morphogenetic Protein (BMP) pathway at the Drosophila larval neuromuscular junction results in nuclear accumulation of the phosphorylated form of the transcription factor Mad in the motoneuron nucleus. This in turn regulates transcription of genes that control synaptic growth. How BMP signaling at the synaptic terminal is relayed to the cell body and nucleus of the motoneuron to regulate transcription is unknown. We show that the BMP receptors are endocytosed at the synaptic terminal and transported retrogradely along the axon. Furthermore, this transport is dependent on BMP pathway activity, as it decreases in the absence of ligand or receptors. We further demonstrate that receptor traffic is severely impaired when Dynein motors are inhibited, a condition that has previously been shown to block BMP pathway activation. In contrast to these results, we find no evidence for transport of phosphorylated Mad along the axons, and axonal traffic of Mad is not affected in mutants defective in BMP signaling or retrograde transport. These data support a model in which complexes of activated BMP receptors are actively transported along the axon towards the cell body to relay the synaptogenic signal, and that phosphorylated Mad at the synaptic terminal and cell body represent two distinct molecular populations.

Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila.

Bidirectional axonal transport driven by kinesin and dynein along microtubules is critical to neuronal viability and function. To evaluate axonal transport mechanisms, we developed a high-resolution imaging system to track the movement of amyloid precursor protein (APP) vesicles in Drosophila segmental nerve axons. Computational analyses of a large number of moving vesicles in defined genetic backgrounds with partial reduction or overexpression of motor proteins enabled us to test with high precision existing and new models of motor activity and coordination in vivo. We discovered several previously unknown features of vesicle movement, including a surprising dependence of anterograde APP vesicle movement velocity on the amount of kinesin-1. This finding is largely incompatible with the biophysical properties of kinesin-1 derived from in vitro analyses. Our data also suggest kinesin-1 and cytoplasmic dynein motors assemble in stable mixtures on APP vesicles and their direction and velocity are controlled at least in part by dynein intermediate chain.

Spectrin mutations that cause spinocerebellar ataxia type 5 impair axonal transport and induce neurodegeneration in Drosophila.

Spinocerebellar ataxia type 5 (SCA5) is an autosomal dominant neurodegenerative disorder caused by mutations in the SPBTN2 gene encoding beta-III-spectrin. To investigate the molecular basis of SCA5, we established a series of transgenic Drosophila models that express human beta-III-spectrin or fly beta-spectrin proteins containing SCA5 mutations. Expression of the SCA5 mutant spectrin in the eye causes a progressive neurodegenerative phenotype, and expression in larval neurons results in posterior paralysis, reduced synaptic terminal growth, and axonal transport deficits. These phenotypes are genetically enhanced by both dynein and dynactin loss-of-function mutations. In summary, we demonstrate that SCA5 mutant spectrin causes adult-onset neurodegeneration in the fly eye and disrupts fundamental intracellular transport processes that are likely to contribute to this progressive neurodegenerative disease.

Asunder is a critical regulator of dynein-dynactin localization during Drosophila spermatogenesis.

Spermatogenesis uses mitotic and meiotic cell cycles coordinated with growth and differentiation programs to generate functional sperm. Our analysis of a Drosophila mutant has revealed that asunder (asun), which encodes a conserved protein, is an essential regulator of spermatogenesis. asun spermatocytes arrest during prophase of meiosis I. Strikingly, arrested spermatocytes contain free centrosomes that fail to stably associate with the nucleus. Spermatocytes that overcome arrest exhibit severe defects in meiotic spindle assembly, chromosome segregation, and cytokinesis. Furthermore, the centriole-derived basal body is detached from the nucleus in asun postmeiotic spermatids, resulting in abnormalities later in spermatogenesis. We find that asun spermatocytes and spermatids exhibit drastic reduction of perinuclear dynein-dynactin, a microtubule motor complex. We propose a model in which asun coordinates spermatogenesis by promoting dynein-dynactin recruitment to the nuclear surface, a poorly understood process required for nucleus-centrosome coupling at M phase entry and fidelity of meiotic divisions.

Dynein light intermediate chain: an essential subunit that contributes to spindle checkpoint inactivation.

The dynein light intermediate chain (LIC) is a subunit unique to the cytoplasmic form of dynein, but how it contributes to dynein function is not fully understood. Previous work has established that the LIC homodimer binds directly to the dynein heavy chain and may mediate the attachment of dynein to centrosomes and other cargoes. Here, we report our characterization of the LIC in Drosophila. Unlike vertebrates, in which two Lic genes encode multiple subunit isoforms, the Drosophila LIC is encoded by a single gene. We determined that the single LIC polypeptide is phosphorylated, and that different phosphoisoforms can assemble into the dynein motor complex. Our mutational analyses demonstrate that, similar to other dynein subunits, the Drosophila LIC is required for zygotic development, germline specification of the oocyte, and mitotic cell division. We show that RNA interference depletion of LIC in Drosophila S2 cells does not block the recruitment of a dynein complex to kinetochores, but it does delay inactivation of Mad2 signaling and mitotic progression. Our observations suggest the LIC contributes to a broad range of dynein functions.

Dynein and Star interact in EGFR signaling and ligand trafficking.

Intracellular transport and processing of ligands is critical to the activation of signal transduction pathways that guide development. Star is an essential gene in Drosophila that has been implicated in the trafficking of ligands for epidermal growth factor (EGF) receptor signaling. The role of cytoplasmic motors in the endocytic and secretory pathways is well known, but the specific requirement of motors in EGF receptor transport has not been investigated. We identified Star in a screen designed to recover second-site modifiers of the dominant rough eye phenotype of the Glued mutation Gl(1). The Glued (Gl) locus encodes the p150 subunit of the dynactin complex, an activator of cytoplasmic dynein-driven motility. We show that alleles of Gl and dynein genetically interact with both Star and EGFR alleles. Similarly to mutations in Star, the Gl(1) mutation is capable of modifying the phenotypes of the EGFR mutation Ellipse. These genetic interactions suggest a model in which Star, dynactin and dynein cooperate in the trafficking of EGF ligands. In support of this model, overexpression of the cleaved, active Spitz ligand can partially bypass defective trafficking and suppress the genetic interactions. Our direct observations of live S2 cells show that export of Spitz-GFP from the endoplasmic reticulum, as well as the trafficking of Spitz-GFP vesicles, depends on both Star and dynein.

Motility screen identifies Drosophila IGF-II mRNA-binding protein--zipcode-binding protein acting in oogenesis and synaptogenesis.

The localization of specific mRNAs can establish local protein gradients that generate and control the development of cellular asymmetries. While all evidence underscores the importance of the cytoskeleton in the transport and localization of RNAs, we have limited knowledge of how these events are regulated. Using a visual screen for motile proteins in a collection of GFP protein trap lines, we identified the Drosophila IGF-II mRNA-binding protein (Imp), an ortholog of Xenopus Vg1 RNA binding protein and chicken zipcode-binding protein. In Drosophila, Imp is part of a large, RNase-sensitive complex that is enriched in two polarized cell types, the developing oocyte and the neuron. Using time-lapse confocal microscopy, we establish that both dynein and kinesin contribute to the transport of GFP-Imp particles, and that regulation of transport in egg chambers appears to differ from that in neurons. In Drosophila, loss-of-function Imp mutations are zygotic lethal, and mutants die late as pharate adults. Imp has a function in Drosophila oogenesis that is not essential, as well as functions that are essential during embryogenesis and later development. Germline clones of Imp mutations do not block maternal mRNA localization or oocyte development, but overexpression of a specific Imp isoform disrupts dorsal/ventral polarity. We report here that loss-of-function Imp mutations, as well as Imp overexpression, can alter synaptic terminal growth. Our data show that Imp is transported to the neuromuscular junction, where it may modulate the translation of mRNA targets. In oocytes, where Imp function is not essential, we implicate a specific Imp domain in the establishment of dorsoventral polarity.

Dynein-mediated apical localization of crumbs transcripts is required for Crumbs activity in epithelial polarity.

Asymmetrical localization of transcripts coupled with localized translation constitutes an important mechanism widely deployed to regulate gene activity in a spatial manner. The conserved transmembrane protein Crumbs (Crb) is an important regulator of epithelial polarity. However, it remains unclear how Crb is targeted to the apical domain. Here, we show that the cytoplasmic dynein complex transports both Crb protein and transcripts to the apical domain of Drosophila melanogaster follicular cells (FCs). The crb 3' untranslated region (UTR) is necessary and sufficient for the apical localization of its transcript and this apical transcript localization is crucial for crb function. In crb mutant FCs, Crb protein derived from transgenes lacking the 3' UTR does not effectively localize to the apical domain and does not effectively restore normal epithelial polarity. We propose that dynein-mediated messenger RNA transport coupled with a localized translation mechanism is involved in localizing Crb to the apical domain to mediate epithelial apicobasal polarity and that this mechanism might be widely used to regulate cellular polarity.

The concentration of Nuf, a Rab11 effector, at the microtubule-organizing center is cell cycle regulated, dynein-dependent, and coincides with furrow formation.

Animal cytokinesis relies on membrane addition as well as acto-myosin-based constriction. Recycling endosome (RE)-derived vesicles are a key source of this membrane. Rab11, a small GTPase associated with the RE and involved in vesicle targeting, is required for elongation of the cytokinetic furrow. In the early Drosophila embryo, Nuclear-fallout (Nuf), a Rab11 effector, promotes vesicle-mediated membrane delivery and actin organization at the invaginating furrow. Although Rab11 maintains a relatively constant localization at the microtubule-organizing center (MTOC), Nuf is present at the MTOC only during the phases of the cell cycle in which furrow invagination occurs. We demonstrate that Nuf protein levels remain relatively constant throughout the cell cycle, suggesting that Nuf is undergoing cycles of concentration and dispersion from the MTOC. Microtubules, but not microfilaments, are required for proper MTOC localization of Nuf and Rab11. The MTOC localization of Nuf also relies on Dynein. Immunoprecipitation experiments demonstrate that Nuf and Dynein physically interact. In accord with these findings, and in contrast to previous reports, we demonstrate that microtubules are required for proper metaphase furrow formation. We propose that the cell cycle-regulated, Dynein-dependent recruitment of Nuf to the MTOC influences the timing of RE-based vesicle delivery to the invaginating furrows.