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Cells are the smallest building blocks of all living eukaryotic organisms, usually ranging from a couple of micrometers (for example, platelets) to hundreds of micrometers (for example, neurons and oocytes) in size. In eukaryotic cells that are more than 100 µm in diameter, very often a self-organized large-scale movement of cytoplasmic contents, known as cytoplasmic streaming, occurs to compensate for the physical constraints of large cells. In this Review, we discuss cytoplasmic streaming in multiple cell types and the mechanisms driving this event. We particularly focus on the molecular motors responsible for cytoplasmic movements and the biological roles of cytoplasmic streaming in cells. Finally, we describe bulk intercellular flow that transports cytoplasmic materials to the oocyte from its sister germline cells to drive rapid oocyte growth., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

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.

Image correlation microscopy methodology was extended and used to determine retrospectively the density, dynamics and interactions of alpha5-integrin in migrating cells. Alpha5-integrin is present in submicroscopic clusters containing 3-4 integrins before it is discernibly organized. The integrin in nascent adhesions, as identified by the presence of paxillin, is approximately 1.4 times more concentrated, approximately 4.5 times more clustered and much less mobile than in surrounding regions. Thus, while integrins are clustered throughout the cell, they differ in nascent adhesions and appear to initiate adhesion formation, despite their lack of visible organization. In more mature adhesions where the integrin is visibly organized there are approximately 900 integrins microm(-2) (about fivefold higher than surrounding regions). Interestingly, alpha5-integrin and alpha-actinin, but not paxillin, reside in a complex throughout the cell, where they diffuse and flow together, even in regions where they are not organized. During adhesion disassembly some integrins diffuse away slowly, alpha-actinin undergoes a directed movement at speeds similar to actin retrograde flow (0.29 microm min(-1)), while all of the paxillin diffuses away rapidly.

The growing pollen tube provides an excellent single cell model system in which to study the mechanisms determining growth regulation, polarity and periodic behaviour. Previously, using FM4-64, we identified periodic movements within the apical vesicle accumulation that were related to the period of oscillatory growth. This suggested a more complex interdependence between membrane traffic, apical extension and periodicity than previously thought. To investigate this a comparison was made between normally growing and Brefeldin-A-treated, non-growing, tubes. Brefeldin-A treatment established an intriguing, stable yet dynamic system of membrane aggregations in the pollen tube tip that exhibited regular movements of material with a 5-7 second period compared with the normal approximately 30 second periodicity observed in growing tubes. Heat treatment was found to reduce period length in both cases. After BFA treatment membrane was demonstrated to flow from the extreme pollen tube apex back through a distinct subapical Brefeldin-A-induced membrane accumulation. The effects of Brefeldin-A on the distribution of ER- and Golgi-targeted fluorescent proteins revealed that ER did not contribute directly to the system of membrane aggregations while only certain compartments of the Golgi might be involved. The involvement of membrane derived from the apical vesicle accumulation was strongly implicated. Calcium measurements revealed that Brefeldin-A abolished the typical tip-focused calcium gradient associated with growth and there were no obvious periodic fluctuations in apical calcium associated with the continued periodic Brefeldin-A membrane aggregation associated movements. Our experiments reveal an underlying periodicity in the pollen tube that is independent of secretion, apical extension and the oscillating tip-focused calcium gradient normally associated with growth, but requires an active actin cytoskeleton.

The highly elongated bristles of Drosophila have proven to be a valuable model system for studying cellular morphogenesis. Extending bristles contain a series of large bundles of actin filaments juxtaposed to the plasma membrane and centrally located microtubules. Models to explain the extension of the bristle have principally focused on the assembly of actin filaments at the distal tip of the bristle. We have used time-lapse observations of wild-type and mutant bristles and the related arista laterals and come to the conclusion that growth takes place throughout the growing cellular extension. This distributed growth can explain the behavior of split laterals and the shape changes seen at the tip during bristle and lateral outgrowth. Inhibitor studies suggest that the microtubule cytoskeleton is essential for maintaining the highly biased axial growth of these structures. We have used fluorescence recovery after photo-bleaching to study the dynamics of the cytoskeleton during bristle growth. Our experiments show that actin bundles in growing bristles are quite stable and move in a retrograde fashion. The bristle microtubules are less stable. The retrograde movement of the peripheral actin appears to be counterbalanced by the distally directed movement of cytoplasm in the center of the bristle.

Cytasters were induced in the unfertilized eggs of the sea urchin Lytechinus pictus by two different methods: (1) treatment with hexylene glycol or taxol, which are known to lower the tubulin critical concentration in vitro, but do not activate the nuclear cycle, and (2) by raising the cytoplasmic pH from that of unfertilized cytoplasm to that of fertilized, which activated the nuclear cycle and initiated tubulin polymerization. In the unactivated eggs, with increasing concentrations of the inducing agents, temperature and duration of treatment, microtubule structures that formed showed a progression from loose microtubule networks and spiral arrays to tightly organized cytasters. In eggs incubated in sea water containing 2.5 or 10 mM ammonium acetate titrated with HCl or NaOH in steps from pH 5.0 to pH 9.0, the nuclear cycle and tubulin polymerization were initiated at about pH 7.0. The degree of development attainable after three hours was dependent on the pH, with spirals forming at the threshold level of pH 7.0, monasters at pH 7.5, and at pH 8.5 cells formed cytasters, multipolar spindles and even completed multipolar divisions. In unactivated eggs, tubulin was made available by lowering of the tubulin critical concentration; in activated eggs it was made available from some previously unavailable store. The evidence suggests that the amount of assembly-competent tubulin available, regardless of how it is made available, determines the type of structure that is formed, with microtubule bundles and spirals at the lower concentrations and functional cytasters at the upper. We also describe some details of cytaster formation, including the role of microtubule interactions and the movement of dense granules to the aster centers in hexylene glycol-treated eggs.