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Fast axonal transport is crucial for neuronal function and is driven by kinesins and cytoplasmic dynein. Here, we investigated the role of kinesin-1 in dense core vesicle (DCV) transport in C. elegans, using mutants in the kinesin light chains (klc-1 and klc-2) and the motor subunit (unc-116) expressing an ida-1::gfp transgene that labels DCVs. DCV transport in both directions was greatly impaired in an unc-116 mutant and had reduced velocity in a klc-2 mutant. In contrast, the speed of retrograde DCV transport was increased in a klc-1 mutant whereas anterograde transport was unaffected. We identified striking differences between the klc mutants in their effects on worm locomotion and responses to drugs affecting neuromuscular junction activity. We also determined lifespan, finding that unc-116 mutant was short-lived whereas the klc single mutant lifespan was wild type. The ida-1::gfp transgenic strain was also short-lived, but surprisingly, klc-1 and klc-2 extended the ida-1::gfp lifespan beyond that of wild type. Our findings suggest that kinesin-1 not only influences anterograde and retrograde DCV transport but is also involved in regulating lifespan and locomotion, with the two kinesin light chains playing distinct roles., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Endosomal sorting complexes required for transport (ESCRT)-0 sorts ubiquitylated EGFR within the early endosome so that the receptor can be incorporated into intralumenal vesicles. An important question is whether ESCRT-0 acts solely upon EGFR that has already entered the vacuolar early endosome (characterised by the presence of EEA1) or engages EGFR within earlier compartments. Here, we employ a suite of software to determine the localisation of ESCRT-0 at subpixel resolution and to perform particle-based colocalisation analysis with other endocytic markers. We demonstrate that although some of the ESCRT-0 subunit Hrs (also known as HGS) colocalises with the vacuolar early endosome marker EEA1, most localises to a population of peripheral EEA1-negative endosomes that act as intermediates in transporting EGFR from the cell surface to more central early endosomes. The peripheral Hrs-labelled endosomes are distinct from APPL1-containing endosomes, but co-label with the novel endocytic adaptor SNX15. In contrast to ESCRT-0, ESCRT-I is recruited to EGF-containing endosomes at later times as they move to more a central position, whereas ESCRT-III is also recruited more gradually. RNA silencing experiments show that both ESCRT-0 and ESCRT-I are important for the transit of EGF to EEA1 endosomes., (© 2015. Published by The Company of Biologists Ltd.)

LIS1, NDE1 and NDEL1 modulate cytoplasmic dynein function in several cellular contexts. However, evidence that they regulate dynein-dependent organelle positioning is limited. Here, we show that depletion of NDE1 or NDEL1 alone profoundly affected the organisation of the Golgi complex but did not cause it to disperse, and slightly affected the position of endocytic compartments. However, striking dispersal of organelles was observed when both NDE1 and NDEL1 were depleted. A substantial portion of NDE1 and NDEL1 is membrane associated, and depletion of these proteins led to complete loss of dynein from membranes. Knockdown of LIS1 also caused the Golgi complex to fragment and disperse throughout the cell, and caused endocytic compartments to relocalise to the periphery. Depletion of LIS1, which is primarily cytosolic, led to partial loss of membrane-associated dynein, without affecting NDE1 and NDEL1. These data suggest that NDE1 and NDEL1 act upstream of LIS1 in dynein recruitment, and/or activation, on the membrane. Consistent with this hypothesis, expression of exogenous NDE1 or NDEL1 rescued the effects of LIS1 depletion on Golgi organisation, whereas LIS1 was only partially effective at rescuing the loss of NDE1 and NDEL1.

Generating the extended endoplasmic reticulum (ER) network depends on microtubules, which act as tracks for motor-driven ER tubule movement, generate the force to extend ER tubules by means of attachment to growing microtubule plus-ends and provide static attachment points. We have analysed ER dynamics in living VERO cells and find that most ER tubule extension is driven by microtubule motors. Surprisingly, we observe that approximately 50% of rapid ER tubule movements occur in the direction of the centre of the cell, driven by cytoplasmic dynein. Inhibition of this movement leads to an accumulation of lamellar ER in the cell periphery. By expressing dominant-negative kinesin-1 constructs, we show that kinesin-1 drives ER tubule extension towards the cell periphery and that this motility is dependent on the KLC1B kinesin light chain splice form but not on KLC1D. Inhibition of kinesin-1 promotes a shift from tubular to lamellar morphology and slows down the recovery of the ER network after microtubule depolymerisation and regrowth. These observations reconcile previous conflicting studies of kinesin-1 function in ER motility in vivo. Furthermore, our data reveal that cytoplasmic dynein plays a role in ER motility in a mammalian cultured cell, demonstrating that ER motility is more complex than previously thought.

Cenp-F is an unusual kinetochore protein in that it localizes to the nuclear matrix in interphase and the nuclear envelope at the G2/M transition; it is farnesylated and rapidly degraded after mitosis. We have recently shown that farnesylation of Cenp-F is required for G2/M progression, its localization to kinetochores, and its degradation. However, the role Cenp-F plays in mitosis has remained enigmatic. Here we show that, following repression of Cenp-F by RNA interference (RNAi), the processes of metaphase chromosome alignment, anaphase chromosome segregation and cytokinesis all fail. Although kinetochores attach to microtubules in Cenp-F-deficient cells, the oscillatory movements that normally occur following K-fibre formation are severely dampened. Consistently, inter-kinetochore distances are reduced. In addition, merotelic associations are observed, suggesting that whereas kinetochores can attach microtubules in the absence of Cenp-F, resolving inappropriate interactions is inhibited. Repression of Cenp-F does not appear to compromise the spindle checkpoint. Rather, the chromosome alignment defect induced by Cenp-F RNA interference is accompanied by a prolonged mitosis, indicating checkpoint activation. Indeed, the prolonged mitosis induced by Cenp-F RNAi is dependent on the spindle checkpoint kinase BubR1. Surprisingly, chromosomes in Cenp-F-deficient cells frequently show a premature loss of chromatid cohesion. Thus, in addition to regulating kinetochore-microtubule interactions, Cenp-F might be required to protect centromeric cohesion prior to anaphase commitment. Intriguingly, whereas most of the sister-less kinetochores cluster near the spindle poles, some align at the spindle equator, possibly through merotelic or lateral orientations.

Plasma membrane blebbing is a defining characteristic of apoptosis, but its significance is not understood. Using live-cell imaging we have identified two phases of apoptotic blebbing. The early phase is restricted to adherent cells, and is prevented by the Rho-activated kinase inhibitor Y27632. The late phase is partially resistant to Y27632, and generates morphologically distinct membrane protrusions that are likely precursors to apoptotic bodies. Late blebbing is observed in all apoptotic cells tested. It occurs at a fixed period before phosphatidyl serine exposure, indicating that it is a universal and important feature of apoptosis. Late blebs contain a cortical layer of endoplasmic reticulum that often surrounds condensed chromatin, while other organelles are excluded. The appearance in some apoptotic cells of partially formed sheets of endoplasmic reticulum suggest that these cortical layers are newly formed by the remodelling of the endoplasmic reticulum of interphase cells. Formation of endoplasmic reticulum and chromatin-containing blebs requires both actin and microtubules, and is prevented by the caspase-6 inhibitor zVEID.fmk.

While the subcellular organisation of mitochondria is likely to influence many aspects of cell physiology, its molecular control is poorly understood. Here, we have investigated the role of the retrograde motor protein complex, dynein-dynactin, in mitochondrial localisation and morphology. Disruption of dynein function, achieved in HeLa cells either by over-expressing the dynactin subunit, dynamitin (p50), or by microinjection of an anti-dynein intermediate chain antibody, resulted in (a) the redistribution of mitochondria to the nuclear periphery, and (b) the formation of long and highly branched mitochondrial structures. Suggesting that an alteration in the balance between mitochondrial fission and fusion may be involved in both of these changes, overexpression of p50 induced the translocation of the fission factor dynamin-related protein (Drp1) from mitochondrial membranes to the cytosol and microsomes. Moreover, a dominant-negative-acting form of Drp1 mimicked the effects of p50 on mitochondrial morphology, while wild-type Drp1 almost completely restored normal mitochondrial distribution in p50 over-expressing cells. Thus, the dynein/dynactin complex plays an unexpected role in the regulation of mitochondrial morphology in living cells, by controlling the recruitment of Drp1 to these organelles.

We report the caspase-dependent cleavage of two Golgi-associated transport factors during apoptosis. The tethering factor giantin is rapidly cleaved both in vitro and in vivo at a conserved site, to generate a stable membrane-anchored domain and a soluble domain that is subject to further caspase-dependent cleavage. The t-SNARE syntaxin 5 is also cleaved rapidly, resulting in the separation of the catalytic membrane-proximal domain from an N-terminal regulatory domain. Cleavage of giantin and syntaxin 5 is accompanied by a cessation of vesicular transport between the ER and the Golgi complex, which first manifests itself as a block in ER exit. The contribution that such an inhibition of trafficking may make towards the generation of an apoptotic phenotype is discussed.

Recruitment of secretory vesicles to the cell surface is essential for the sustained secretion of insulin in response to glucose. At present, the molecular motors involved in this movement, and the mechanisms whereby they may be regulated, are undefined. To investigate the role of kinesin family members, we labelled densecore vesicles in clonal beta-cells using an adenovirally expressed, vesicle-targeted green fluorescent protein (phogrin.EGFP), and employed immunoadsorption to obtain highly purified insulin-containing vesicles. Whereas several kinesin family members were expressed in this cell type, only conventional kinesin heavy chain (KHC) was detected in vesicle preparations. Expression of a dominant-negative KHC motor domain (KHC(mut)) blocked all vesicular movements with velocity >0.4 micro m second(-1), which demonstrates that kinesin activity was essential for vesicle motility in live beta-cells. Moreover, expression of KHC(mut) strongly inhibited the sustained, but not acute, stimulation of secretion by glucose. Finally, vesicle movement was stimulated by ATP dose-dependently in permeabilized cells, which suggests that glucose-induced increases in cytosolic [ATP] mediate the effects of the sugar in vivo, by enhancing kinesin activity. These data therefore provide evidence for a novel mechanism whereby glucose may enhance insulin release.