ESCRT-III-associated proteins and spastin inhibit protrudin-dependent polarised membrane traffic.

Mutations in the gene encoding the microtubule severing ATPase spastin are the most frequent cause of hereditary spastic paraplegia, a genetic condition characterised by length-dependent axonal degeneration. Here, we show that HeLa cells lacking spastin and embryonic fibroblasts from a spastin knock-in mouse model become highly polarised and develop cellular protrusions. In HeLa cells, this phenotype was rescued by wild-type spastin, but not by forms unable to sever microtubules or interact with endosomal ESCRT-III proteins. Cells lacking the spastin-interacting ESCRT-III-associated proteins IST1 or CHMP1B also developed protrusions. The protrusion phenotype required protrudin, a RAB-interacting protein that interacts with spastin and localises to ER-endosome contact sites, where it promotes KIF5-dependent endosomal motility to protrusions. Consistent with this, the protrusion phenotype in cells lacking spastin also required KIF5. Lack or mutation of spastin resulted in functional consequences for receptor traffic of a pathway implicated in HSP, as Bone Morphogenetic Protein receptor distribution became polarised. Our results, therefore, identify a novel role for ESCRT-III proteins and spastin in regulating polarised membrane traffic.

ER-export and ARFRP1/AP-1-dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication.

The ß-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release, and pathogenesis. We developed a nondisruptive tagging strategy for SARS-CoV-2 E and find that, at steady state, it localizes to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for endoplasmic reticulum-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP ribosylation factor 1/adaptor protein-1 (ARFRP1/AP-1)-dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across ß-coronaviruses, and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and, by developing a trans-complementation assay for SARS-CoV-2 structural proteins, that lysosomal delivery of E and its viroporin activity is necessary for efficient viral replication and release.

Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia.

Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER-endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell-derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER-endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration.

Chirp-pulse-compression three-dimensional lidar imager with fiber optics.

A coherent three-dimensional (angle-angle-range) lidar imager using a master-oscillator-power-amplifier concept and operating at a wavelength of 1.5 microm with chirp-pulse compression is described. A fiber-optic delay line in the local oscillator path enables a single continuous-wave semiconductor laser source with a modulated drive waveform to generate both the constant-frequency local oscillator and the frequency chirp. A portion of this chirp is gated out and amplified by a two-stage fiber amplifier. The digitized return signal was compressed by cross correlating it with a sample of the outgoing pulse. In this way a 350-ns, 10-microJ pulse with a 250-MHz frequency sweep is compressed to a width of approximately 8 ns. With a 25-mm output aperture, the lidar has been used to produce three-dimensional images of hard targets out to a range of approximately 2 km with near-diffraction-limited angular resolution and submeter range resolution.

Analysis of the performance of a coherent pulsed fiber lidar for aerosol backscatter applications.

The antenna and the Doppler estimation characteristics of a coherent pulsed lidar intended for short-range aerosol backscatter applications have been analyzed. The system used fiber-optic interconnects and operated at a wavelength of 1.548 microm. The range dependence of the signal for various bistatic and monostatic antenna configurations has been determined. The system operated in a low-pulse-energy, high-pulse-repetition-rate mode, and the Doppler estimates from the return signal were achieved with a multipulse accumulation procedure. The expected performance of the accumulation in this low-photocount regime was compared with the data obtained from the system, and a reasonable level of agreement was demonstrated.

Remote photoacoustic detection of liquid contamination of a surface.

A method for the remote detection and identification of liquid chemicals at ranges of tens of meters is presented. The technique uses pulsed indirect photoacoustic spectroscopy in the 10-microm wavelength region. Enhanced sensitivity is brought about by three main system developments: (1) increased laser-pulse energy (150 microJ/pulse), leading to increased strength of the generated photoacoustic signal; (2) increased microphone sensitivity and improved directionality by the use of a 60-cm-diameter parabolic dish; and (3) signal processing that allows improved discrimination of the signal from noise levels through prior knowledge of the pulse shape and pulse-repetition frequency. The practical aspects of applying the technique in a field environment are briefly examined, and possible applications of this technique are discussed.

High-energy in-fiber pulse amplification for coherent lidar applications.

An Er:Yb codoped fiber amplifier chain for the generation of pulses for coherent lidar applications at a wavelength near 1.5 microm is reported. The final 1.8-m-long power amplification stage had a 50-microm core diameter and yielded a 23-dB energy gain, resulting in 0.29-mJ, 100-ns pulses at a repetition rate of 4 kHz with no Brillouin scattering and an M2 of 2.1.