Since the earliest examination of cellular structures, biologists have been fascinated by observing cells using light microscopy. The advent of fluorescent labeling technologies plus the plethora of sophisticated light microscope techniques now available make studying dynamic processes in living cells almost commonplace. For anyone new to this area, however, it can be daunting to decide which techniques or equipment to try. Here, we aim to give a brief overview of the main approaches to live cell imaging, with some mention of their pros and cons.

Mitochondria display a variety of shapes, ranging from small and spherical or the classical tubular shape to extended networks. Shape transitions occur frequently and include fusion, fission, and branching. It was reported that some mitochondrial shape transitions are developmentally regulated, whereas others were linked to disease or apoptosis. However, if and how mitochondrial function controls mitochondrial shape through regulation of mitochondrial fission and fusion is unclear. Here, we show that inhibitors of electron transport, ATP synthase, or the permeability transition pore (mtPTP) induced reversible mitochondrial fission. Mitochondrial fission depended on dynamin-related protein 1 (DRP1) and F-actin: Disruption of F-actin attenuated fission and recruitment of DRP1 to mitochondria. In contrast, uncoupling of electron transport and oxidative phosphorylation caused mitochondria to adopt a distinct disk shape. This shape change was independent of the cytoskeleton and DRP1 and was most likely caused by swelling. Thus, disruption of mitochondrial function rapidly and reversibly altered mitochondrial shape either by activation of DRP1-dependent fission or by swelling, indicating a close relationship between mitochondrial fission, shape, and function. Furthermore, our results suggest that the actin cytoskeleton is involved in mitochondrial fission by facilitating mitochondrial recruitment of DRP1.

he mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved T specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.

HighlightsTransport between endocytic organelles involves microtubule and actin based molecular motors.Both cytoskeletal systems contribute to membrane deformation and scission.Transport of endosomes to and from the cell cortex requires transfer between actin and microtubules.

After viral fusion, capsids of the neurotropic herpes simplex virus are transported along microtubules (MT) to the nuclear pores for viral genome uncoating, nuclear transcription and replication. After assembly and egress from the nucleus, cytosolic capsids are transported to host membranes for secondary envelopment or to the axon terminal for further viral spread. Using GFP-tagged capsids, Cy3-labelled MT and cytosol, we have reconstituted viral capsid transport in vitro. In the presence of ATP, capsids moved along MT up to 30 mm. Blocking the function of dynactin, a cofactor of dynein and kinesin-2, inhibited the transport. Removing outer tegument proteins from the capsids increased in vitro motility. In contrast, capsids isolated from infected nuclei that were devoid of inner as well as outer tegument proteins showed little interaction with dynein and its cofactor dynactin. Our data suggest that the inner tegument of alphaherpesviruses contains viral receptors for MT motors. Herpes simplex virus type 1 (HSV1) is a neurotropic human alphaherpesvirus that initially infects the oral or perioral skin and mucosa before amplified virus enters local sensory and autonomic nerve endings (1). An HSV1 virion consists of a DNA-containing capsid that is covered by about 20 different capsid-associated and tegument proteins, and capsid and tegument are enveloped by a viral membrane (2). Most likely, capsids lose their envelope before moving to the neuronal cell bodies located in cranial ganglia (3-5). The viral dsDNA genome of 152 kb is injected into the nucleoplasm through the nuclear pore (6) and establishes a lifelong latent infection. Upon reactivation, progeny capsids, and possibly virions, contained in membrane vesicles are transported anterogradely to the peripheral nerve endings (7-12). In rare cases, reactivated virus is instead transported to the central nervous system causing life-threatening encephalitis (13).It has been predicted that it would take an HSV1 capsid with a diameter of 125 nm 231 years to diffuse 10 mm in the axonal cytoplasm (14). Instead of diffusion, viral particles use the host cytoskeleton for fast intracellular transport (1,15,16). Microtubules (MT) are long cytoskeletal filaments with biochemically distinct ends assembled from a/b-tubulin (17). The fast-growing plus-ends of MT usually point towards the plasma membrane and in neuronal axons towards the nerve terminals. The lessdynamic MT minus-ends are often stabilized by attachment to a MT-organizing centre located close to the nucleus. HSV1 loses its envelope during cell entry by fusion with the plasma membrane or with an endocytic membrane (18,19), and in epithelial as well as in neuronal cells, incoming capsids, possibly with associated tegument proteins, are transported along MT to the nucleus (20-23). Efficient virus assembly and egress also depend on MT (24,25), and progeny virus uses MT for efficient axonal transport to the synapse (11).Cytoplasmic dynein together with its cofactor dynactin powers most transport to MT minus-ends (26),...