Kinesin and dynein mutants provide novel insights into the roles of vesicle traffic during cell morphogenesis in Neurospora.

BACKGROUND: Kinesin and cytoplasmic dynein are force-generating molecules that move in opposite directions along microtubules. They have been implicated in the directed transport of a wide variety of cellular organelles, but it is unclear whether they have overlapping or largely independent functions. RESULTS: We analyzed organelle transport in kinesin and dynein single mutants, and in a kinesin and dynein double mutant of Neurospora crassa. Remarkably, the simultaneous mutation of kinesin and dynein was not lethal and resulted in an additive phenotype that combined the features of the single mutants. The mutation of kinesin and dynein had opposite effects on the apical and retrograde transport, respectively, of vesicular organelles. In the kinesin mutant, apical movement of submicroscopic, secretory vesicles to the Spitzenk?rper - an organelle in the hyphal apex - was defective, whereas the predominantly retrograde movement of microscopic organelles was only slightly reduced. In contrast, the dynein mutant still had a prominent Spitzenk?rper, demonstrating that apical transport was intact, but retrograde transport was essentially inhibited completely. A major defect in vacuole formation and dynamics was also evident. In agreement with the observations on apical transport, protein secretion into the medium was markedly inhibited in the kinesin mutant but not in the dynein mutant. CONCLUSIONS: Transport of secretory vesicles is necessary but not sufficient for normal apical extension. A component of retrograde transport, presumably precursors of the vacuole system, is also essential. Our findings provide new information on the role microtubule motors play in cell morphogenesis and suggest that kinesin and cytoplasmic dynein have largely independent functions within separate pathways.     

Association of kinesin with characterized membrane-bounded organelles.

The family of molecular motors known as kinesin has been implicated in the translocation of membrane-bounded organelles along microtubules, but relatively little is known about the interaction of kinesin with organelles. In order to understand these interactions, we have examined the association of kinesin with a variety of organelles. Kinesin was detected in purified organelle fractions, including synaptic vesicles, mitochondria, and coated vesicles, using quantitative immunoblots and immunoelectron microscopy. In contrast, isolated Golgi membranes and nuclear fractions did not contain detectable levels of kinesin. These results demonstrate that the organelle binding capacity of kinesin is selective and specific. The ability to purify membrane-bounded organelles with associated kinesin indicates that at least a portion of the cellular kinesin has a relatively stable association with membrane-bounded organelles in the cell. In addition, immunoelectron microscopy of mitochondria revealed a patch-like pattern in the kinesin distribution, suggesting that the organization of the motor on the organelle membrane may play a role in regulating organelle motility.     

Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport

The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.     

Microtubule motors and pollen tube growth—still an open question

The growth of pollen tubes is supported by the continuous supply of secretory vesicles in the tip area. Movement and accumulation of vesicles is driven by the dynamic interplay between the actin cytoskeleton and motor proteins of the myosin family. A combination of the two protein systems is also responsible for the bidirectional movement of larger organelle classes. In contrast, the role of microtubules and microtubule-based motors is less clear and often ambiguous. Nevertheless, there is evidence which shows that the pollen tube contains a number of microtubule-based motors of the kinesin family. These motor proteins are likely to be associated with pollen tube organelles and, consequently, they have been hypothesized to participate in the distribution of organelles during pollen tube growth. Whether microtubule motor proteins take part in either the transport or positioning of organelles is not known for sure, but there is evidence for this second possibility. This review will discuss the current knowledge of microtubule-based motor proteins (including kinesins and hypothetical dyneins) and will make some hypothesis about their role in the pollen tube.     

Cytoplasmic motors and pollen tube growth

The growth of pollen tubes is characterized by an intense cytoplasmic streaming, during which the movements of smaller organelles (like secretory vesicles) and larger ones (including the generative cell and vegetative nucleus) are precisely coordinated. A well-characterized cytoskeletal apparatus is likely responsible for these intracellular movements. In recent years both microfilament and microtubule-based motor proteins have been identified and assumed to be the translocators of the several organelle categories. Their precise function during pollen tube growth is not yet clear, but apparently an actomyosin-based system is mainly responsible for pollen tube elongation. On the other hand, microtubules and microtubule-based motors have been thought to play a role in the maintenance of cell polarity. Both cytoskeletal systems (and their respective motor activities) could cooperate to ensure a precise regulation of pollen tube growth.     

Targeting of secretory vesicles to cytoplasmic domains in AtT-20 and PC- 12 cells

Organelles are not uniformly distributed throughout the cytoplasm but have preferred locations that vary between tissues and during development. To investigate organelle targeting to cytoplasmic domains we have taken advantage of the mouse pituitary cell line, AtT-20, which, when induced to extend long processes, accumulates dense core secretory granules at the tips of the processes. During mitosis, these secretory granules accumulate along the plane of division. Protein synthesis is not mandatory for such redistribution of secretory granules. To explore the specificity of the redistribution we have used transfected AtT-20 cells that express the immunoglobulin kappa light chain. While the endogenous hormone ACTH is found in secretory granules, the kappa chain is a marker for organelles involved in constitutive secretion. By immunofluorescence, kappa also accumulates at the tips of growing processes, and along the midline of dividing cells, suggesting that the redistribution of vesicles is not specific for dense-core secretory granules. Since there is evidence for selective organelle transport along processes in neuronal cells, the rat pheochromocytoma cell PC-12 was transfected with DNA encoding markers for regulated and constitutive secretory vesicles. Again regulated and constitutive vesicles co-distribute, even in cells grown in the presence of nerve growth factor. We suggest that at least in the cells studied here, cytoskeletal elements normally carry exocytotic organelles to the surface; when the cytoskeletal elements coalesce in an extending process, exocytotic organelles of both the constitutive and regulated pathway are transported nonselectively to the tips of the cytoskeletal elements where they accumulate.     

Kinesin- and Myosin-driven Steps of Vesicle Recruitment for Ca2+-regulated Exocytosis

Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca²⁺-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca²⁺/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin– dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca²⁺-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.     

The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenk?rper in Neurospora crassa

Most models for fungal growth have proposed a directional traffic of secretory vesicles to the hyphal apex, where they temporarily aggregate at the Spitzenkörper before they fuse with the plasma membrane (PM). The PM H⁺-translocating ATPase (PMA-1) is delivered via the classical secretory pathway (endoplasmic reticulum [ER] to Golgi) to the cell surface, where it pumps H⁺ out of the cell, generating a large electrochemical gradient that supplies energy to H⁺-coupled nutrient uptake systems. To characterize the traffic and delivery of PMA-1 during hyphal elongation, we have analyzed by laser scanning confocal microscopy (LSCM) strains of Neurospora crassa expressing green fluorescent protein (GFP)-tagged versions of the protein. In conidia, PMA-1-GFP was evenly distributed at the PM. During germination and germ tube elongation, PMA-1-GFP was found all around the conidial PM and extended to the germ tube PM, but fluorescence was less intense or almost absent at the tip. Together, the data indicate that the electrochemical gradient driving apical nutrient uptake is generated from early developmental stages. In mature hyphae, PMA-1-GFP localized at the PM at distal regions (>120 μm) and in completely developed septa, but not at the tip, indicative of a distinct secretory route independent of the Spitzenkörper occurring behind the apex.     

Kinesin motors as molecular machines

Molecular motor proteins, fueled by energy from ATP hydrolysis, move along actin filaments or microtubules, performing work in the cell. The kinesin microtubule motors transport vesicles or organelles, assemble bipolar spindles or depolymerize microtubules, functioning in basic cellular processes. The mechanism by which motor proteins convert energy from ATP hydrolysis into work is likely to differ in basic ways from man-made machines. Several mechanical elements of the kinesin motors have now been tentatively identified, permitting researchers to begin to decipher the mechanism of motor function. The force-producing conformational changes of the motor and the means by which they are amplified are probably different for the plus- and minus-end kinesin motors.     

Kinectin participates in microtubule-dependent hormone secretion in pancreatic islet beta-cells

Kinectin (KNT) is a candidate membrane receptor for kinesin in the movement of intracellular organelles along microtubules. Isoforms of KNT exist containing different combinations of six small (residues 23-33) variable domains (vd) vd1-6 within the C-terminus. Here we investigate a role for KNT and its isoform KNTvd4(-) in the transport of amylin and insulin-containing secretory vesicles in the pancreatic islet beta-cell line RINm5F. KNTvd4(-) lacks vd4 that forms the kinesin-binding domain, and hence its role in the cell is an enigma. We report that amylin-containing vesicles also contained insulin, and exhibited microtubule, and small G-protein-dependent secretion. Knockdown of KNT by small interference RNA (siRNA) inhibited amylin expression and secretion. In contrast, recombinant KNTvd4(-) overexpressed in RINm5F cells associated with amylin-containing vesicles and inhibited amylin secretion, but had no discernible affect on amylin expression. The data suggests that both KNT and KNTvd4(-) participate in microtubule-dependent secretion of amylin in islet beta-cells.