Lysosomal uptake of isolated cell organelles microinjected into HeLa cells

Lysosomes and microsomes were isolated from rat liver and microinjected into the cytoplasm of HeLa cells. The fate of the transplanted organelles and their effects on the recipient cells were followed in the electron microscope at various time intervals after administration. Needle injection with buffer or sucrose did not seem to evoke any ultrastructural alterations, such as induced autophagy or other signs of sublethal cell injury. Recipients of microinjected cell organelles elicited a rapid and conspicuous increase in membrane-bounded cytoplasmic vacuoles, concomitant with the disappearance of the injected material. Golgi complexes became abundant with many small vesicles clustering around their cisternae. The volume density of the lysosomal compartment increased 2-3-fold after organelle injection as compared with control-injected (0.3 M sucrose) or noninjected cells. Our preliminary results show that isolated cell organelles can be microinjected into cells n culture and indicate that the microinjected organelles were segregated from the cytoplasm into membrane-bounded vacuoles probably through autophagolysosome formation. Thus, this technique offers an additional approach for studies on the segregation and degradation of cell organelles in somatic cells and may enable more detailed analyses on the mechanisms of autophagic sequestration of specific cell organelles.     

Degradation of structurally characterized proteins injected into HeLa cells. Effects of intracellular location and the involvement of lysosomes

Thirty-five proteins of known x-ray structure were radioiodinated and injected into HeLa cells. The cells were then cultured in the presence or absence of the lysosomotropic agents, ammonium chloride and chloroquine. These compounds did not inhibit the degradation of an injected protein unless its half-life was greater than 45 h. Among the more stable proteins the extent of inhibition was proportional to their half-lives. These results indicate that all injected proteins are transferred to lysosomes at comparable rates such that the fraction of a specific protein degraded in lysosomes depends upon its rate of degradation in the cytosol. That is, basal autophagy is nonselective in HeLa cells. The intracellular location of each injected protein was measured by homogenization of injected cells in sucrose and differential sedimentation or by extraction in buffers containing Triton X-100. Solubilities of the injected proteins ranged from 6 to 89%, and stabilities of 10 proteins, originally extracellular in function, were inversely proportional to their solubility. These results illustrate the potential importance of subcellular location on protein stability in the cytosol.     

Microinjection studies of protein transit across the nuclear envelope of human cells

Proteins of various molecular weights were conjugated with rhodamine and microinjected into the cytoplasm or nucleus of HeLa cells. The injected proteins were then localized within the cells at various times thereafter with fluorescence microscopy. Proteins below approx. 60 kD rapidly crossed the HeLa nuclear envelope. Some larger proteins also were able to pass into or out of the nucleus, while others were unable to do so, indicating the selective permeability of the HeLa nuclear envelope to large proteins. The nuclear protein HMG17 accumulated within the nucleus shortly after cytoplasmic microinjection.     

Comparative studies on microinjected high-mobility-group chromosomal proteins, HMG1 and HMG2

The nonhistone chromosomal proteins, HMG1 and HMG2, were iodinated and introduced into HeLa cells, bovine fibroblasts, or mouse 3T3 cells by erythrocyte-mediated microinjection. Autoradiographic analysis of injected cells fixed with glutaraldehyde consistently showed both molecules concentrated within nuclei. Fixation with methanol, on the other hand, resulted in some leakage of the microinjected proteins from the nuclei so that more autoradiographic grains appeared over the cytoplasm or outside the cells. Both injected and endogenous HMG1 and HMG2 partitioned unexpectedly upon fractionation of bovine fibroblasts, HeLa, or 3T3 cells, appearing in the cytoplasmic fractions. However, in calf thymus, HMG1 and HMG2 molecules appeared in the 0.35 M NaCl extract of isolated nuclei, as expected. These observations show that the binding of HMG1 and HMG2 to chromatin differs among cell types or that other tissue-specific components can influence their binding. Coinjection of [125I]HMG1 and [131I]HMG2 into HeLa cells revealed that the two molecules display virtually equivalent distributions upon cell fractionation, identical stability, identical intracellular distributions, and equal rates of equilibration between nuclei. In addition, HMG1 and HMG2 did not differ in their partitioning upon fractionation nor in their stability in growing vs. nongrowing 3T3 cells. Thus, we have not detected any significant differences in the intracellular behavior of HMG1 and HMG2 after microinjection into human, bovine, or murine cells.     

Degradation of erythrocyte-microinjected and scrape-loaded homologous cytosolic proteins by 3T3-L1 cells.

Homologous cytosol was introduced into 3T3-L1 cells by two different methods. Erythrocytes loaded with radiolabelled cytosolic proteins extracted from 3T3-L1 cells were fused with the aid of Sendai virus to 3T3-L1 cells, which were then seeded to confluent and non-confluent cultures. Cytosolic proteins were also introduced into cells by the technique of scrape-loading. In confluent cells, injected cytosolic proteins were recovered largely (54-93%) in a sedimentable (6 X 10(6) gav.-min) fraction from recipient cells irrespective of the method of introduction or of radiolabelling of the injected proteins [( 125I]iodination, reductive methylation with NaB3H4 and backbone labelling with L-[4,5-3H]leucine). The degradation of microinjected cytosolic proteins was in all cases inhibited by the lysosomotropic agent NH4Cl to a greater extent (32-75%) than that observed for endogenous cytosolic (less than or equal to 19%) proteins (labelled with L-[4,5-3H]leucine). In growing cells both endogenous total cell proteins and microinjected proteins were degraded at a slower rate than in confluent cell monolayers. The inhibition by NH4Cl of the degradation of both the endogenous and microinjected proteins is decreased compared with the inhibition observed in confluent monolayers. The results are discussed in terms of the cytoplasmic capacity to segregate microinjected homologous proteins before protein degradation can take place.     

Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.     

Protein migration into nuclei. I. Frog oocyte nuclei in vivo accumulate microinjected histones, allow entry to small proteins, and exclude large proteins

A technique is presented which enables one to measure the extent to which a protein enters and accumulates in the nucleus of the frog oocyte. In this method, the protein, labeled with 125-I, is microinjected into the oocyte. After incubation, the oocyte is manually enucleated and the radioactivity in the nucleus and cytoplasm is determined. Using this technique, proteins lighter than 20,000 daltons were found to enter the nucleus and completely equilibrate between the nucleus and cytoplasm within 24 h. The entry of proteins heavier than 69,000 daltons was severely hindered. Histones and histone fractions entered as quickly as other small proteins, but, in contrast to these proteins, they accumulated in the nucleus to different extents, depending on the total amount of histone injected into the oocyte and the identity of the histone. Evidence is presented that histone fractions compete with each other for accumulation in the nucleus.     

Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993). We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [3H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides.     

Intracellular distribution of Tankyrases as detected by multicolor immunofluorescence techniques

Poly(ADP-ribose) polymerases are a family of enzymes that catalyze the conversion of NAD+ into ADP-ribose. Among them, Tankyrases have been found to bind to centrosome, mitotic spindle and microsome proteins, in the cytoplasm, and to telomeres in the nucleus, where they play a relevant role in telomere metabolism. However, their precise intracellular localization during interphase has not been so far fully elucidated. We investigated this aspect in situ by double immunofluorescence experiments using antibodies recognizing Tankyrases 1-2 or other proteins residing in specific organelles (Golgi apparatus, mitochondria, lysosomes, endoplasmic reticulum). We used HeLa cells as a model system in vitro, before and after treatment with either actinomycin D or etoposide, to also investigate the possible relocation of Tankyrases during apoptosis. We observed that Tankyrases are distributed both in the nucleus and in the cytoplasm; in this latter compartment, they were found to colocate with the Golgi apparatus but never with the mitochondria; a pool of Tankyrases also colocates with the endoplasmic reticulum and lysosomes. Interestingly, in cells with clear signs of apoptosis, Tankyrases were detectable in the cytoplasmic blebs: this suggests that they are not massively cleaved during apoptosis and persist in the largely heterogeneous apoptotic remnants which are known to contain components of cytoplasmic and nuclear origin.     

Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants

Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide.     

Microinjection of intermediate filament proteins into living cells with and without preexisting intermediate filament network

Human cells grown in monolayer culture were microinjected with intermediate filament subunit proteins. In fibroblasts with a preexisting vimentin network, injected porcine glial fibrillary acidic protein (GFAP) co-localized with the vimentin network within 24 hours. Phosphorylated GFAP variants were found to become dephosphorylated concomitantly with their incorporation into filamentous structures. After microinjection of either porcine GFAP or murine vimentin into human carcinoma cells lacking cytoplasmic intermediate filaments, we observed that different types of filament networks developed. Whereas vimentin was incorporated into short filaments immediately after injection, GFAP was found to aggregate into rodlike structures. This may indicate a differential filament forming ability of these intermediate filament proteins in vivo.     

Targeting of lysosomal integral membrane protein LIMP II. The tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient for direct targeting to lysosomes.

Time course experiments of the localization of rat LIMP II expressed in COS cells show that the protein is transported directly from the Golgi complex to lysosomes. Substitution of the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II for the native cytoplasmic tails of the plasma membrane proteins CD36 and CD8 resulted in straight transport of both proteins to lysosomes. The synthetic tyrosine-containing heptapeptide, RGTGVYG, did not replace the natural carboxyl cytoplasmic tail of LIMP II in its ability to transport both CD36 and CD8 to lysosomes, and the two constructs were transported to and expressed at the plasma membrane. Substitution of the cytoplasmic tails of either CD36 or CD8 for the carboxyl cytoplasmic tail of LIMP II resulted in transport of the mutants to the plasma membrane where they underwent endocytosis before accumulating into lysosomes. The results indicate that a motif contained in the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II is sufficient to target proteins directly from the Golgi complex to lysosomes.     

Orthopoxvirus gene expression in Xenopus laevis oocytes: a component of the virion is needed for late gene expression.

We have examined the feasibility of using Xenopus laevis oocytes microinjected with rabbit poxvirus as a system to study poxvirus gene expression. The injection of either intact virus or subviral cores resulted in accurate synthesis of viral proteins. This expression was dependent on the multiplicity of injected virus, with the optimal injected dose being equivalent to approximately 300 PFU per oocyte. Extensive viral gene expression including late viral protein synthesis was observed when intact virions were microinjected into the oocyte. However, the injection of subviral cores resulted in only early protein synthesis. When oocytes were injected with a mixture of subviral cores and the nonionic detergent-soluble fraction was removed from virus during the preparation of cores, both early and late viral proteins were synthesized. Therefore, the detergent-soluble fraction appears to contain a factor(s) required for the transition from early to late gene expression.     

Age-related change in the degradation rate of ovalbumin microinjected into mouse liver parenchymal cells

Change in the degradation rate of ovalbumin (OVA) microinjected into liver parenchymal cells isolated from mice of various ages was studied. OVA was injected by osmotic lysis of pinosomes and the amount of OVA was determined by immunoblotting using purified antibody to OVA. Cellular activity as judged by rates of protein synthesis and degradation of pulse-labeled proteins was not affected by the injection. To localize injected OVA in the cells, cell extracts were fractionated by differential centrifugation and the amount of OVA and the activity of beta-D-galactosidase as a lysosomal marker enzyme of each fraction were determined. The ovalbumin was mostly found in the soluble fraction, while the beta-D-galactosidase activity was found in the particulate fraction, indicating that the ovalbumin was spread in the cytosol but was not present in the pinosomes or lysosomes. The average half-lives of OVA were 106, 113, and 164 h in the cells from young (3.5-6.5 months), middle-aged (13.5-20.0 months), and old (24.5-30.5 months) mice, respectively. Thus, the half-life of ovalbumin in the cells of senescent mice was about 50% longer than that in the cells of young or middle-aged mice. These results are in good agreement with those of our previous investigation, which showed that the half-life of inactivation of horseradish peroxidase was extended by about 50% in the hepatocytes from old mice (Ishigami and Goto, 1988, Mech. Ageing Dev., 46, 125-133).     

Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells

Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA-gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted.     

10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin

Cells were microinjected with four mouse monoclonal antibodies that were directed against either alpha- or beta-tubulin subunits, one monoclonal with activity against both subunits, and a guinea pig polyclonal antibody with activity directed against both subunits, to determine the effects on the distribution of cytoplasmic microtubules and 10-nm filaments. The specificities of the antibodies were confirmed by Western blots, solid phase radioimmunoassay, and Western blot analysis of alpha- and beta-tubulin peptide maps. Two monoclonals DM1A and DM3B3, an anti-alpha- and anti-beta-tubulin respectively, and the guinea pig polyclonal anti-alpha/beta-tubulin antibody (GP1T4) caused the 10-nm filaments to collapse into large lateral aggregates collecting in the cell periphery or tight juxtanuclear caps; the other monoclonal antibodies had no effect when microinjected into cells. The filament collapsing was observed to be complete at 1.5-2 h after injection. During the first 30 min after injection a few cytoplasmic microtubules near the cell periphery could be observed by fluorescence microscopy. This observation was confirmed by electron microscopy, which also demonstrated assembled microtubules in the juxtanuclear region. By 1.5 h, when most of the 10-nm filaments were collapsed, the complete cytoplasmic array of microtubules was observed. Cells injected in prophase were able to assemble a mitotic spindle, suggesting that the antibody did not block microtubule assembly. Metabolic labeling with [35S]methionine of microinjected cells revealed that total protein synthesis was the same as that observed in uninjected cells. This indicated that the microinjected antibody apparently did not produce deleterious effects on cellular metabolism. These results suggest that through a direct interaction of antibodies with either alpha- or beta- tubulin subunits, 10-nm filaments were dissociated from their normal distribution. It is possible that the antibodies disrupted postulated 10-nm filament-microtubule interactions.     

Antibodies against the chemically synthesized genome-linked protein of poliovirus react with native virus-specific proteins

The genome-linked protein (VPg) of poliovirus has been chemically synthesized, coupled to bovine serum albumin carrier and injected into rabbits. An antibody response was elicited not only by the full-length synthetic VPg peptide, but also by a synthetic 14-amino acid carboxy-terminal peptide. All antisera reacted with virus-specific proteins from HeLa cells infected with poliovirus. Three of these proteins have previously been implicated by others as precursors of VPg. No free cytoplasmic VPg could be detected, and the antibodies did not react with radiolabeled proteins from uninfected cells.     

HeLa DNA polymerase alpha activity in vitro: specific stimulation by a non-enzymic protein factor.

A non-enzymic protein factor that increases the in vitro rate of synthesis by HeLa DNA polymerase alpha 15- to 30-fold with denatured DNA as template has been partially purified from the cytoplasmic fraction of HeLa cells. The stimulatory effect is highly specific for HeLa DNA polymerase alpha and for DNA templates that contain extensive regions of single-strandedness. Synthesis with denatured DNA as template presumably proceeds from 3'-hydroxyl termini formed at loop-back regions since the synthesized DNA product and template are covalently linked. The stimulatory protein factor chromatographs as a basic protein, has an approximate molecular weight of 30,000 daltons and binds with moderate affinity to denatured DNA cellulose, being eluted by o.4M NaCl. The purified factor lacks detectable DNA polymerase, exo- and endodeoxyribonuclease and RNA polymerase activities. It also does not promote helix-coil transitions with poly[d(A-T)] and Clostridium perfringens DNA.